A comprehensive dataset of protein-protein interactions and ligand binding pockets for advancing drug discovery.

This dataset represents a collection of pocket-centric structural data related to protein-protein interactions (PPIs) and PPI-related ligand binding sites. The dataset includes high-quality structural information on more than 23,000 pockets, 3,700 proteins on more than 500 organisms, and nearly 3500 ligands that can aid researchers in the fields of bioinformatics, structural biology, and drug discovery. It encompasses a diverse set of PPI complexes with more than 1,700 unique protein families including some with associated ligands, enabling detailed investigations into molecular interactions at the atomic level. This article introduces an indispensable resource designed to unlock the full potential of PPIs while pioneering a novel metric for pocket similarity for hypothesizing protein partners repurposing.

Uncovering domain motif interactions using high-throughput protein-protein interaction detection methods.

Protein-protein interactions (PPIs) are often mediated by short linear motifs (SLiMs) in one protein and domain in another, known as domain-motif interactions (DMIs). During the past decade, SLiMs have been studied to find their role in cellular functions such as post-translational modifications, regulatory processes, protein scaffolding, cell cycle progression, cell adhesion, cell signalling and substrate selection for proteasomal degradation. This review provides a comprehensive overview of the current PPI detection techniques and resources, focusing on their relevance to capturing interactions mediated by SLiMs. We also address the challenges associated with capturing DMIs. Moreover, a case study analysing the BioGrid database as a source of DMI prediction revealed significant known DMI enrichment in different PPI detection methods. Overall, it can be said that current high-throughput PPI detection methods can be a reliable source for predicting DMIs.

The C-terminal protein interaction domain of the chromatin reader Yaf9 is critical for pathogenesis of Candida albicans.

Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10 000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Recording physiological history of cells with chemical labeling.

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

Interaction of human dendritic cell receptor DEC205/CD205 with keratins.

DEC205 (CD205) is one of the major endocytic receptors on dendritic cells and has been widely used as a receptor target in immune therapies. It has been shown that DEC205 can recognize dead cells through keratins in a pH-dependent manner. However, the mechanism underlying the interaction between DEC205 and keratins remains unclear. Here we determine the crystal structures of an N-terminal fragment of human DEC205 (CysR∼CTLD3). The structural data show that DEC205 shares similar overall features with the other mannose receptor family members such as the mannose receptor and Endo180, but the individual domains of DEC205 in the crystal structure exhibit distinct structural features that may lead to specific ligand binding properties of the molecule. Among them, CTLD3 of DEC205 adopts a unique fold of CTLD, which may correlate with the binding of keratins. Furthermore, we examine the interaction of DEC205 with keratins by mutagenesis and biochemical assays based on the structural information and identify an XGGGX motif on keratins that can be recognized by DEC205, thereby providing insights into the interaction between DEC205 and keratins. Overall, these findings not only improve the understanding of the diverse ligand specificities of the mannose receptor family members at the molecular level but may also give clues for the interactions of keratins with their binding partners in the corresponding pathways.

Molecular insights into atypical modes of ß-arrestin interaction with seven transmembrane receptors.

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.

Molecular Basis for SPINDOC-Spindlin1 Engagement and Its Role in Transcriptional Attenuation.

Spindlin1 is a histone reader with three Tudor-like domains and its transcriptional co-activator activity could be attenuated by SPINDOC. The first two Tudors are involved in histone methylation readout, while the function of Tudor 3 is largely unknown. Here our structural and binding studies revealed an engagement mode of SPINDOC-Spindlin1, in which a hydrophobic motif of SPINDOC, DOCpep3, stably interacts with Spindlin1 Tudor 3, and two neighboring K/R-rich motifs, DOCpep1 and DOCpep2, bind to the acidic surface of Spindlin1 Tudor 2. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPINDOC fragment containing the K/R-rich region attenuates histone or TCF4 binding by Spindlin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 "K4me3-K9me3" bivalent mark. Further ChIP-seq and RT-qPCR indicated that SPINDOC could promote genomic relocation of Spindlin1, thus modulate downstream gene transcription. Collectively, we revealed multivalent engagement between SPINDOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPINDOC-Spindlin1 association, while K/R-rich region modulates the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activity of Spindlin1.

HOCOMOCO in 2024: a rebuild of the curated collection of binding models for human and mouse transcription factors.

We present a major update of the HOCOMOCO collection that provides DNA binding specificity patterns of 949 human transcription factors and 720 mouse orthologs. To make this release, we performed motif discovery in peak sets that originated from 14 183 ChIP-Seq experiments and reads from 2554 HT-SELEX experiments yielding more than 400 thousand candidate motifs. The candidate motifs were annotated according to their similarity to known motifs and the hierarchy of DNA-binding domains of the respective transcription factors. Next, the motifs underwent human expert curation to stratify distinct motif subtypes and remove non-informative patterns and common artifacts. Finally, the curated subset of 100 thousand motifs was supplied to the automated benchmarking to select the best-performing motifs for each transcription factor. The resulting HOCOMOCO v12 core collection contains 1443 verified position weight matrices, including distinct subtypes of DNA binding motifs for particular transcription factors. In addition to the core collection, HOCOMOCO v12 provides motif sets optimized for the recognition of binding sites in vivo and in vitro, and for annotation of regulatory sequence variants. HOCOMOCO is available at https://hocomoco12.autosome.org and https://hocomoco.autosome.org.

Intrinsically Disordered Regions Control cBAF Condensation and Interactions.

Intrinsically disordered regions (IDR) of ARID1A/B control cBAF condensation and protein-protein interactions.

Comprehensive comparative assessment of the Arabidopsis thaliana MLO2-CALMODULIN2 interaction by various in vitro and in vivo protein-protein interaction assays.

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.

Mechanistic Insights into the Interactions of Arl8b with the RUN Domains of PLEKHM1 and SKIP.

Arl8b, a specific Arf-like family GTPase present on lysosome, and plays critical roles in many lysosome-related cellular processes such as autophagy. The active Arl8b can be specifically recognized by the RUN domains of two Arl8b-effectors PLEKHM1 and SKIP, thereby regulating the autophagosome/lysosome membrane fusion and the intracellular lysosome positioning, respectively. However, the mechanistic bases underlying the interactions of Arl8b with the RUN domains of PLEKHM1 and SKIP remain elusive. Here, we report the two high-resolution crystal structures of the active Arl8b in complex with the RUN domains of PLEKHM1 and SKIP. In addition to elucidating the detailed molecular mechanism governing the specific interactions of the active Arl8b with the RUN domains of PLEKHM1 and SKIP, the determined complex structures also reveal a general binding mode shared by the PLEKHM1 and SKIP RUN domains for interacting with the active Arl8b. Furthermore, we uncovered a competitive relationship between the RUN domains of PLEKHM1 and SKIP in binding to the active Arl8b as well as a unique small GTPase-binding mode adopted by the PLEKHM1 and SKIP RUN domains, thereby enriching the repertoire of the RUN domain/small GTPase interaction modes. In all, our findings provide new mechanistic insights into the interactions of the active Arl8b with PLEKHM1 and SKIP, and are valuable for further understanding the working modes of these proteins in relevant cellular processes.

Revealing Allostery in PTPN11 SH2 Domains from MD Simulations.

Src-homology 2 (SH2) domains are protein interaction domains that bind to specific peptide motifs containing phosphotyrosine. SHP2, a tyrosine phosphatase encoded by PTPN11 gene, which has been emerged as positive or negative modulator in multiple signaling pathways, contains two SH2 domains, respectively, called N-SH2 and C-SH2. These domains play a relevant role in regulating SHP2 activity, either by recognizing its binding partners or by blocking its catalytic site. Considering the multiple functions that these domains carry out in SHP2, N-SH2 and C-SH2 represent an interesting case of study. In this chapter, we present a methodology that permits, by means of the principal component analysis (PCA), to study and to rationalize the structures adopted by the SH2 domains, in terms of the conformations of their binding sites. The structures can be distinguished, grouped, classified, and reported in a diagram. This approach permits to identify the accessible conformations of the SH2 domains in different binding conditions and to eventually reveal allosteric interactions. The method further reveals that the conformation dynamics of N-SH2 and C-SH2 strongly differ, which likely reflects their distinct functional roles.

Lipid Binding of SH2 Domains.

The Src homology 2 (SH2) domain is a modular protein interaction domain that specifically recognizes the phosphotyrosine (pY) motif of a target molecule. We recently reported that a large majority of human SH2 domains tightly bind membrane lipids, and many show high lipid specificity. Most of them can bind a lipid and the pY motif coincidently because their lipid-binding sites are topologically distinct from pY-binding pockets. Lipid binding of SH2 domain-containing kinases and phosphatases is functionally important because it exerts exquisite spatiotemporal control on protein-protein interaction and cell signaling activities mediated by these proteins. Here, we describe two assays, surface plasmon resonance analysis and fluorescence quenching analysis, which allow quantitative determination of the affinity and specificity of SH2-lipid interaction and high-throughput screening for SH2 domain-lipid-binding inhibitors.

Combined prediction and design reveals the target recognition mechanism of an intrinsically disordered protein interaction domain.

An increasing number of protein interaction domains and their targets are being found to be intrinsically disordered proteins (IDPs). The corresponding target recognition mechanisms are mostly elusive because of challenges in performing detailed structural analysis of highly dynamic IDP-IDP complexes. Here, we show that by combining recently developed computational approaches with experiments, the structure of the complex between the intrinsically disordered C-terminal domain (CTD) of protein 4.1G and its target IDP region in NuMA can be dissected at high resolution. First, we carry out systematic mutational scanning using dihydrofolate reductase-based protein complementarity analysis to identify essential interaction regions and key residues. The results are found to be highly consistent with an α/ß-type complex structure predicted by AlphaFold2 (AF2). We then design mutants based on the predicted structure using a deep learning protein sequence design method. The solved crystal structure of one mutant presents the same core structure as predicted by AF2. Further computational prediction and experimental assessment indicate that the well-defined core structure is conserved across complexes of 4.1G CTD with other potential targets. Thus, we reveal that an intrinsically disordered protein interaction domain uses an α/ß-type structure module formed through synergistic folding to recognize broad IDP targets. Moreover, we show that computational prediction and experiment can be jointly applied to segregate true IDP regions from the core structural domains of IDP-IDP complexes and to uncover the structure-dependent mechanisms of some otherwise elusive IDP-IDP interactions.

Cooperative regulation of C1-domain membrane recruitment polarizes atypical protein kinase C.

Recruitment of the Par complex protein atypical protein kinase C (aPKC) to a specific membrane domain is a key step in the polarization of animal cells. While numerous proteins and phospholipids interact with aPKC, how these interactions cooperate to control its membrane recruitment has been unknown. Here, we identify aPKC's C1 domain as a phospholipid interaction module that targets aPKC to the membrane of Drosophila neural stem cells (NSCs). The isolated C1 binds the NSC membrane in an unpolarized manner during interphase and mitosis and is uniquely sufficient among aPKC domains for targeting. Other domains, including the catalytic module and those that bind the upstream regulators Par-6 and Bazooka, restrict C1's membrane targeting activity-spatially and temporally-to the apical NSC membrane during mitosis. Our results suggest that aPKC polarity results from cooperative activation of autoinhibited C1-mediated membrane binding activity.

Structural basis of γ chain family receptor sharing at the membrane level.

Common γ chain (γc) cytokine receptors, including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with a common γc receptor (CD132) by concomitant binding of their ectodomains to an interleukin. In this work, we find that direct interactions between the transmembrane domains (TMDs) of both the γc and the interleukin receptors (ILRs) are also required for receptor activation. Moreover, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences within the family. Heterodimer structures of γc TMD bound to IL-7 and IL-9 receptor TMDs-determined in a lipid bilayer-like environment by nuclear magnetic resonance spectroscopy-reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Thus, signaling in the γc receptor family requires specific heterotypic interactions of the TMDs.

Deploying synthetic coevolution and machine learning to engineer protein-protein interactions.

Fine-tuning of protein-protein interactions occurs naturally through coevolution, but this process is difficult to recapitulate in the laboratory. We describe a platform for synthetic protein-protein coevolution that can isolate matched pairs of interacting muteins from complex libraries. This large dataset of coevolved complexes drove a systems-level analysis of molecular recognition between Z domain-affibody pairs spanning a wide range of structures, affinities, cross-reactivities, and orthogonalities, and captured a broad spectrum of coevolutionary networks. Furthermore, we harnessed pretrained protein language models to expand, in silico, the amino acid diversity of our coevolution screen, predicting remodeled interfaces beyond the reach of the experimental library. The integration of these approaches provides a means of simulating protein coevolution and generating protein complexes with diverse molecular recognition properties for biotechnology and synthetic biology.

7TM domain structures of adhesion GPCRs: what's new and what's missing?

Adhesion-type G protein-coupled receptors (aGPCRs) have long resisted approaches to resolve the structural details of their heptahelical transmembrane (7TM) domains. Single-particle cryogenic electron microscopy (cryo-EM) has recently produced aGPCR 7TM domain structures for ADGRD1, ADGRG1, ADGRG2, ADGRG3, ADGRG4, ADGRG5, ADGRF1, and ADGRL3. We review the unique properties, including the position and conformation of their activating tethered agonist (TA) and signaling motifs within the 7TM bundle, that the novel structures have helped to identify. We also discuss questions that the kaleidoscope of novel aGPCR 7TM domain structures have left unanswered. These concern the relative positions, orientations, and interactions of the 7TM and GPCR autoproteolysis-inducing (GAIN) domains with one another. Clarifying their interplay remains an important goal of future structural studies on aGPCRs.

Enzymatic Regulation of Protein-Protein Interactions in Artificial Cells.

Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein-protein or protein-nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein-protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.