Explore the vast range of titles available.

Tight junctions are complex supramolecular entities composed of integral membrane proteins, membrane-associated and soluble cytoplasmic proteins engaging in an intricate and dynamic system of protein–protein interactions. Three-dimensional structures of several tight-junction proteins or their isolated domains have been determined by X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. These structures provide direct insight into molecular interactions that contribute to the formation, integrity, or function of tight junctions. In addition, the known experimental structures have allowed the modeling of ligand-binding events involving tight-junction proteins. Here, we review the published structures of tight-junction proteins. We show that these proteins are composed of a limited set of structural motifs and highlight common types of interactions between tight-junction proteins and their ligands involving these motifs.

A high-throughput mutagenesis study in a PDZ domain shows that biochemical function and adaptation primarily originate from a collectively evolving amino acid network within the structure termed a protein sector. Coevolving sectors make protein design adaptable Statistical analysis of protein evolution suggests a 'design' for natural proteins in which sparse networks of coevolving amino acids comprise the essence of three-dimensional structure and function. To better understand the relationship of sector-based architecture to these properties, the authors performed a comprehensive single-mutation study of a PSD95 pdz3 — a typical PDZ family protein — in which each position is substituted independently of every other amino acid. PDZ domains, which are made up of tens of amino acids, are conserved in many signalling proteins in animals, plants and other organisms. Mutational analysis showed that sector positions are functionally sensitive to mutation, whereas non-sector positions are much more tolerant to substitution, and that adaptation to a new binding specificity initiates exclusively through variation within sector residues. These results show how proteins can be robust yet also capable of rapid functional change when conditions of selection change. Statistical analysis of protein evolution suggests a design for natural proteins in which sparse networks of coevolving amino acids (termed sectors) comprise the essence of three-dimensional structure and function 1 , 2 , 3 , 4 , 5 . However, proteins are also subject to pressures deriving from the dynamics of the evolutionary process itself—the ability to tolerate mutation and to be adaptive to changing selection pressures 6 , 7 , 8 , 9 , 10 . To understand the relationship of the sector architecture to these properties, we developed a high-throughput quantitative method for a comprehensive single-mutation study in which every position is substituted individually to every other amino acid. Using a PDZ domain (PSD95 pdz3 ) model system, we show that sector positions are functionally sensitive to mutation, whereas non-sector positions are more tolerant to substitution. In addition, we find that adaptation to a new binding specificity initiates exclusively through variation within sector residues. A combination of just two sector mutations located near and away from the ligand-binding site suffices to switch the binding specificity of PSD95 pdz3 quantitatively towards a class-switching ligand. The localization of functional constraint and adaptive variation within the sector has important implications for understanding and engineering proteins.

Abstract Some genes encoding proteins within the co-evolved pre- and postsynaptic compartments are present in genomes long preceding the origination of the synapse within the animal kingdom. DLG4, gene encoding PSD-95, is one of the most abundant synaptic proteins. It is a MAGUK family member that shares a conserved domain structure comprised of one or multiple PDZ domains, a Src homology 3 (SH3), and a guanylate kinase (GK) domain. Here, we construct the phylogeny of the tri-PDZ domains in DLG4 to its deep ancestral origin in Filozoa, which includes animals and their nearest unicellular relatives. PDZ domain architecture appears to be a strong organizing feature of this gene lineage that originated with a single ancestral PDZ3-like domain in Capsaspora owczarzaki from which PDZ1 and PDZ2 were derived. The strong conservation of individual PDZ domain identities was captured by Evolutionary Scale Modeling (ESM2) across the boundary to the animal kingdom, corroborating distinct clades formed by the divergence of PDZ1, PDZ2, and PDZ3 in the phylogeny. CRIPT, PDZ3 ligand, is present in all Filozoa genomes studied here. AlphaFold2 Multimer demonstrates conserved binding function; however, conserved binding does not completely depend on either sequence motifs or hydrophobicity profiles. Rather, the most conserved feature is hydrogen bonds at the 0 and −2 positions of the ligand as an ancient foundational innovation for PDZ3 ligand interaction. Hydrogen bonds may loosen the sequence requirements for binding to allow a more extensive search space for protein-protein interactions that enhance fitness before the mutations that secure those interactions occur.

In this study, the authors show that PTEN alters synaptic function after PDZ-dependent recruitment into spines induced by amyloid-β. This mechanism is crucial for pathogenesis, as preventing PTEN-PDZ interactions renders neurons resistant to amyloid-β and rescues cognitive function in Alzheimer's disease models. This suggests that PTEN is a critical effector of the synaptic pathology associated with Alzheimer's disease. Dyshomeostasis of amyloid-β peptide (Aβ) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimer's disease. Aβ appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimer's disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aβ-induced depression. Mechanistically, Aβ triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aβ-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aβ-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aβ signaling.

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain–SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host–virus protein–protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen–host protein–protein interactions.

The high‐risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ‐domain‐containing proteins through its carboxy‐terminal motif. This PDZ‐binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6‐transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRASG12V) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild‐type E6. Interestingly, we found decreased expression of PAR3 in E6‐expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ‐domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRASG12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild‐type E6. These data highlight the importance of the carboxy‐terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes. HPV16 E6 mutant lacking the carboxy‐terminal motif showed markedly reduced tumorigenic ability compared with the wild type. Among putative E6 targets, depletion of MAGI1, SCRIB and PAR3 significantly restored the tumorigenic ability, indicating critical roles of the PDZ‐binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes.

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. Graphical Abstract 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.

PDZ domains are abundant interaction hubs found in a number of different proteins and they exhibit characteristic differences in their structure and ligand specificity. Their internal dynamics have been proposed to contribute to their biological activity via changes in conformational entropy upon ligand binding and allosteric modulation. Here we investigate dynamic structural ensembles of PDZ3 of the postsynaptic protein PSD-95, calculated based on previously published backbone and side-chain S2 order parameters. We show that there are distinct but interdependent structural rearrangements in PDZ3 upon ligand binding and the presence of the intramolecular allosteric modulator helix α3. We have also compared these rearrangements in PDZ1-2 of PSD-95 and the conformational diversity of an extended set of PDZ domains available in the PDB database. We conclude that although the opening-closing rearrangement, occurring upon ligand binding, is likely a general feature for all PDZ domains, the conformer redistribution upon ligand binding along this mode is domain-dependent. Our findings suggest that the structural and functional diversity of PDZ domains is accompanied by a diversity of internal motional modes and their interdependence.

Wnt/β-catenin signaling is hyper-activated in several cancer cells and cancer stem cells. Dishevelled/Dvl is a key adapter protein that acts as a bridge between the Wnt receptor Frizzled (Fzd) and other cytosolic factors. In detail, the C-terminal cytosolic region is the ligand of the PSD-95, disks large, and zonula occludens-1 (PDZ) domain of Dvl. Therefore, the PDZ domain (Dvl-PDZ) is thought to be a potential drug target. In this paper, we determined the first crystal structure of the PDZ domain of human Dvl1 (hDvl1-PDZ) at a 2.4 Å resolution. The domain was adapted into a unique trimeric form in which all the canonical ligand-binding clefts were occupied by the β2-β3 loop of the neighbor molecule, like an auto-inhibiting trimer. We used solution nuclear magnetic resonance (NMR) experiments to assess the presence of the self-associated oligomer of hDvl1-PDZ in the solution. Introducing the Ala substitution at Asp 272, the key residue of the β2-β3 loop, partly abolished the concentration-dependent chemical shift change, which suggests that this residue is one of the key residues for formation. Based on these observations, we propose an auto-inhibiting trimer formation of Dvl-PDZ in a Dvl-Axin hetero-oligomerization model of Wnt/β-catenin signal transduction.

Dynamic allosterism allows the propagation of signal throughout a protein. The PDZ (PSD-95/Dlg1/ZO-1) family has been named as a classic example of dynamic allostery in small modular domains. While the PDZ family consists of more than 200 domains, previous efforts have primarily focused on a few well-studied PDZ domains, including PTP-BL PDZ2, PSD-95 PDZ3, and Par6 PDZ. Taken together, experimental and computational studies have identified regions of these domains that are dynamically coupled to ligand binding. These regions include the αA helix, the αB lower-loop, and the αC helix. In this review, we summarize the specific residues on the αA helix, the αB lower-loop, and the αC helix of PTP-BL PDZ2, PSD-95 PDZ3, and Par6 PDZ that have been identified as participants in dynamic allostery by either experimental or computational approaches. This review can serve as an index for researchers to look back on the previously identified allostery in the PDZ family. Interestingly, our summary of previous work reveals clear consistencies between the domains. While the PDZ family has a low sequence identity, we show that some of the most consistently identified allosteric residues within PTP-BL PDZ2 and PSD-95 PDZ3 domains are evolutionarily conserved. These residues include A46/A347, V61/V362, and L66/L367 on PTP-BL PDZ2 and PSD-95 PDZ3, respectively. Finally, we expose a need for future work to explore dynamic allostery within (1) PDZ domains with multiple binding partners and (2) multidomain constructs containing a PDZ domain.