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Background: PDZ domains are highly abundant protein-protein interaction modules involved in the wiring of protein networks. Emerging evidence indicates that some PDZ domains also interact with phosphoinositides (PtdInsPs), important regulators of cell polarization and signaling. Yet our knowledge on the prevalence, specificity, affinity, and molecular determinants of PDZ-PtdInsPs interactions and on their impact on PDZ-protein interactions is very limited. Methodology/Principal Findings: We screened the human proteome for PtdInsPs interacting PDZ domains by a combination of in vivo cell-localization studies and in vitro dot blot and Surface Plasmon Resonance (SPR) experiments using synthetic lipids and recombinant proteins. We found that PtdInsPs interactions contribute to the cellular distribution of some PDZ domains, intriguingly also in nuclear organelles, and that a significant subgroup of PDZ domains interacts with PtdInsPs with affinities in the low-to-mid micromolar range. In vitro specificity for the head group is low, but with a trend of higher affinities for more phosphorylated PtdInsPs species. Other membrane lipids can assist PtdInsPs-interactions. PtdInsPs-interacting PDZ domains have generally high pI values and contain characteristic clusters of basic residues, hallmarks that may be used to predict additional PtdInsPs interacting PDZ domains. In tripartite binding experiments we established that peptide binding can either compete or cooperate with PtdInsPs binding depending on the combination of ligands. Conclusions/Significance: Our screen substantially expands the set of PtdInsPs interacting PDZ domains, and shows that a full understanding of the biology of PDZ proteins will require a comprehensive insight into the intricate relationships between PDZ domains and their peptide and lipid ligands. [ABSTRACT FROM AUTHOR]

Abstract: The interaction of Na+ and K+ with proteins is at the basis of numerous processes of biological importance. However, measurement of the kinetic components of the interaction has eluded experimentalists for decades because the rate constants are too fast to resolve with conventional stopped-flow methods. Using a continuous-flow apparatus with a dead time of 50 μs we have been able to resolve the kinetic rate constants and entire mechanism of Na+ binding to thrombin, an interaction that is at the basis of the procoagulant and prothrombotic roles of the enzyme in the blood. [Copyright &y& Elsevier]

Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome. Synopsis: Hundreds of mutations in the intrinsically disordered regions of the human proteome that break, diminish, enhance or create motif-based interactions are uncovered by mutational proteomic peptide-phage display. A novel peptide-phage display library that combines disease-associated amino acid changing mutations with the intrinsically disordered regions of the human proteome is described. 275 mutations associated with various diseases are reported to affect 279 motif-based protein-protein interactions. About 50% of the mutations enhance or create binding motifs, and the rest disrupt or diminish interactions. The study provides novel insights into how disease-associated mutations perturb and rewire the motif-based interactome. Hundreds of mutations in the intrinsically disordered regions of the human proteome that break, diminish, enhance or create motif-based interactions are uncovered by mutational proteomic peptide-phage display. [ABSTRACT FROM AUTHOR]

Short linear motifs (SLiMs) are a unique and ubiquitous class of protein interaction modules that perform key regulatory functions and drive dynamic complex formation. For decades, interactions mediated by SLiMs have accumulated through detailed low-throughput experiments. Recent methodological advances have opened this previously underexplored area of the human interactome to high-throughput protein–protein interaction discovery. In this article, we discuss that SLiM-based interactions represent a significant blind spot in the current interactomics data, introduce the key methods that are illuminating the elusive SLiM-mediated interactome of the human cell on a large scale, and discuss the implications for the field. • Tens of thousands of interactions in the human cell mediated by short linear motifs (SLiMs) are predicted to remain undiscovered. • In recent years, several novel experimental approaches for high-throughput motif discovery have been developed. • These methods will allow the mapping of an underexplored part of the human interactome at amino acid resolution. • Several of the methods also allow affinity data to be quantified for a large number of interactions. • Many of the newly characterized interactions will modulate key regulatory processes. [ABSTRACT FROM AUTHOR]

Phosphorylation is a ubiquitous post‐translation modification that regulates protein function by promoting, inhibiting or modulating protein–protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide‐phage display library to screen for phosphosites that modulate short linear motif‐based interactions. The peptidome covers ~13,500 phospho‐serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild‐type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif‐mediated interactions. Affinity measurements confirmed the phospho‐modulation of 14 out of 18 tested interactions. We performed a detailed follow‐up on a phospho‐dependent interaction between clathrin and the mitotic spindle protein hepatoma‐upregulated protein (HURP), demonstrating the essentiality of the phospho‐dependency to the mitotic function of HURP. Structural characterisation of the clathrin‐HURP complex elucidated the molecular basis for the phospho‐dependency. Our work showcases the power of phosphomimetic ProP‐PD to discover novel phospho‐modulated interactions required for cellular function. Synopsis: A new phosphomimetic proteomic peptide phage library is used to screen for phospho‐modulated interactions between short linear motifs and protein domains. Follow‐up analyses show that S839 phosphorylation of HURP is required for interaction with clathrin and mitotic function. A novel phosphomimetic ProP‐PD library is generated, displaying intrinsically disordered regions of the proteome with functionally prioritised phosphosites.Phosphomimetic ProP‐PD selections provide binding preferences for wild‐type, phosphomimetic and phosphorylated peptides at large‐scale.S839 HURP phosphorylation is required for clathrin binding and its role in mitosis.SLiM‐based interactions from ProP‐PD selections are available in a web‐based resource (http://slim.icr.ac.uk/proppd/). [ABSTRACT FROM AUTHOR]

Human mixed lineage leukemia 4 (MLL4), also known as KMT2D, regulates cell type specific transcriptional programs through enhancer activation. Along with the catalytic methyltransferase domain, MLL4 contains seven less characterized plant homeodomain (PHD) fingers. Here, we report that the sixth PHD finger of MLL4 (MLL4 PHD6) binds to the hydrophobic motif of ten-eleven translocation 3 (TET3), a dioxygenase that converts methylated cytosine into oxidized derivatives. The solution NMR structure of the TET3-MLL4 PHD6 complex and binding assays show that, like histone H4 tail, TET3 occupies the hydrophobic site of MLL4 PHD6 , and that this interaction is conserved in the seventh PHD finger of homologous MLL3 (MLL3 PHD7). Analysis of genomic localization of endogenous MLL4 and ectopically expressed TET3 in mouse embryonic stem cells reveals a high degree overlap on active enhancers and suggests a potential functional relationship of MLL4 and TET3. [Display omitted] • PHD6 finger of MLL4 (MLL4 PHD6) binds to ten-eleven translocation 3 (TET3) • TET3 and histone H4K16ac tail occupy the same hydrophobic site of MLL4 PHD6 • Binding to TET3 is conserved in the PHD finger of homologous MLL3 (MLL3 PHD7) • TET3 and MLL4 binding sites overlap on active enhancers in mouse ESCs The human methyltransferase MLL4 and the dioxygenase TET3 mediate fundamental DNA-templated nuclear processes. Becht et al. report the structural mechanism by which MLL4 binds to TET3. [ABSTRACT FROM AUTHOR]

Specific protein–protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein–protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type‐specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide‐phage display library that tiles all disordered regions of the human proteome and allows the screening of ~ 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. Finally, we show how the amino acid resolution‐binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome‐wide discovery of SLiM‐based interactions. Synopsis: An optimized phage peptidome that tiles the disordered regions of the human proteome is presented, allowing the field of motif‐based interactions to transition into high‐throughput. Guidelines and tools for data analysis are provided. An optimized second generation human disorderome (HD2) phage library tiles all disordered regions from the human proteome.Different peptide display parameters are tested, including display on the major or minor coat proteins of the M13 phage, and splitting the library design based sub‐cellular localization of the peptide containing proteins.PepTools is a dedicated toolkit to annotate peptides and to identify consensus motifs.> 2,000 motif‐based interactions are presented, together with information on potential disease mutations or phosphorylation sites that might affect the interactions. [ABSTRACT FROM AUTHOR]

The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes., (© 2024. The Author(s).)

The modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Using proteomic peptide phage display (ProP-PD) coupled with structural, biochemical and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs, including response to nutrient deprivation and glucose metabolism. These findings illustrate a mode of OGT substrate recognition and offer key insights into the biological roles of this unique domain., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (K D ∼ 7-300 μM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways., (© 2023. Springer Nature Limited.)

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics., (© 2023. The Author(s).)

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents., (© 2021. The Author(s).)

PSD95-Disc large-Zonula occludens (PDZ) domains are among the most abundant modular domains in the human proteome. They typically bind short carboxy-terminal sequence motifs of their ligand proteins, which may be transmembrane proteins such as ion channels and GPCRs, as well as soluble proteins. The identity of the endogenous ligands of many PDZ domains remains unclear despite more than two decades of PDZ research. Combinatorial peptide phage display and bioinformatics predictions have contributed to shed light on PDZ-mediated interactions. However, the efficiency of these methods for the identification of interactions of potential biological relevance is hampered by different biases. Proteomic peptide-phage display (ProP-PD) was developed to overcome these limitations. Here we describe a ProP-PD protocol for the identification of C-terminal PDZ domain ligands. The method efficiently identifies peptide ligands within a proteome of interest, and pinpoint targets of potential biological relevance.

The intrinsically disordered regions of the proteome are enriched in short linear motifs (SLiMs) that serve as binding sites for peptide binding proteins. These interactions are often of low-to-mid micromolar affinities and are challenging to screen for experimentally. However, a range of dedicated methods have been developed recently, which open for screening of SLiM-based interactions on large scale. A variant of phage display, termed proteomic peptide phage display (ProP-PD), has proven particularly useful for the purpose. Here, we describe a complete high-throughput ProP-PD protocol for screening intrinsically disordered regions for SLiMs. The protocol requires some basic bioinformatics skills for the design of the library and for data analysis but can be performed in a standard biochemistry lab. The protocol starts from the construction of a library, followed by the high-throughput expression and purification of bait proteins, the phage selection, and the analysis of the binding-enriched phage pools using next-generation sequencing. As the protocol generates rather large data sets, we also emphasize the importance of data management and storage.