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Homer1 is a synaptic scaffold protein that regulates glutamatergic synapses and spine morphogenesis. HOMER1 knockout (KO) mice show behavioral abnormalities related to psychiatric disorders, and HOMER1 has been associated with psychiatric disorders such as addiction, autism disorder (ASD), schizophrenia (SZ), and depression. However, the mechanisms by which it promotes spine stability and its global function in maintaining the synaptic proteome has not yet been fully investigated. Here, we used computational approaches to identify global functions for proteins containing the Homer1-interacting PPXXF motif within the postsynaptic compartment. Ankyrin-G was one of the most topologically important nodes in the postsynaptic peripheral membrane subnetwork, and we show that one of the PPXXF motifs, present in the postsynaptically-enriched 190 kDa isoform of ankyrin-G (ankyrin-G 190), is recognized by the EVH1 domain of Homer1. We use proximity ligation combined with super-resolution microscopy to map the interaction of ankyrin-G and Homer1 to distinct nanodomains within the spine head and correlate them with spine head size. This interaction motif is critical for ankyrin-G 190’s ability to increase spine head size, and for the maintenance of a stable ankyrin-G pool in spines. Intriguingly, lack of Homer1 significantly upregulated the abundance of ankyrin-G, but downregulated Shank3 in cortical crude plasma membrane fractions. In addition, proteomic analysis of the cortex in HOMER1 KO and wild-type (WT) mice revealed a global reshaping of the postsynaptic proteome, surprisingly characterized by extensive upregulation of synaptic proteins. Taken together, we show that Homer1 and its protein interaction motif have broad global functions within synaptic protein-protein interaction networks. Enrichment of disease risk factors within these networks has important implications for neurodevelopmental disorders including bipolar disorder, ASD, and SZ.

The shapes of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by accelerating actin filament assembly locally and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes filopodia assembly and forms part of a poorly understood molecular complex that remains associated with growing filopodia tips. Here we identify precursors of this filopodia tip complex in migrating B16F1 cells: small leading-edge clusters of the adaptor protein lamellipodin (Lpd) that subsequently recruit VASP and initiate filopodia formation. Dimerization, membrane association, and VASP binding are all required for lamellipodin to incorporate into filopodia tip complexes, and overexpression of monomeric, membrane--targeted lamellipodin mutants disrupts tip complex assembly. Once formed, tip complexes containing VASP and lamellipodin grow by fusing with each other, but their growth is limited by a size-dependent dynamic instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between Ena/VASP proteins, EVH1 ligands, and actin filaments.

Metazoan proteomes contain many paralogous proteins that have evolved distinct functions. The Ena/VASP family of actin regulators consists of three members that share an EVH1 interaction domain with a 100% conserved binding site. A proteome-wide screen revealed ciliary protein PCARE as a high-affinity ligand for ENAH EVH1. Here we report the surprising observation that PCARE is ~100-fold specific for ENAH over paralogs VASP and EVL and can selectively bind and inhibit ENAH-dependent adhesion in cells. Specificity arises from a mechanism whereby PCARE stabilizes a conformation of the ENAH EVH1 domain that is inaccessible to family members VASP and EVL. Structure-based modeling rapidly identified seven residues distributed throughout EVL that are sufficient to differentiate binding by ENAH vs. EVL. By exploiting the ENAH-specific conformation, we rationally designed the tightest and most selective ENAH binder to date. Our work uncovers a conformational mechanism of interaction specificity that distinguishes highly similar paralogs and establishes tools for dissecting specific Ena/VASP functions in processes including cancer cell invasion.

Background Dendrite morphogenesis plays an essential role in establishing the connectivity and receptive fields of neurons during the development of the nervous system. To generate the diverse morphologies of branched dendrites, neurons use external cues and cell surface receptors to coordinate intracellular cytoskeletal organization; however, the molecular mechanisms of how this signaling forms branched dendrites are not fully understood. Methods We performed in vivo time-lapse imaging of the PVD neuron in C. elegans in several mutants of actin regulatory proteins, such as the WAVE Regulatory Complex (WRC) and UNC-34 (homolog of Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP)). We examined the direct interaction between the WRC and UNC-34 and analyzed the localization of UNC-34 in vivo using transgenic worms expressing UNC-34 fused to GFP. Results We identify a stereotyped sequence of morphological events during dendrite outgrowth in the PVD neuron in C. elegans. Specifically, local increases in width (“swellings”) give rise to filopodia to facilitate a “rapid growth and pause” mode of growth. In unc-34 mutants, filopodia fail to form but swellings are intact. In WRC mutants, dendrite growth is largely absent, resulting from a lack of both swelling and filopodia formation. We also found that UNC-34 can directly bind to the WRC. Disrupting this binding by deleting the UNC-34 EVH1 domain prevented UNC-34 from localizing to swellings and dendrite tips, resulting in a stunted dendritic arbor and reduced filopodia outgrowth. Conclusions We propose that regulators of branched and linear F-actin cooperate to establish dendritic branches. By combining our work with existing literature, we propose that the dendrite guidance receptor DMA-1 recruits the WRC, which polymerizes branched F-actin to generate “swellings” on a mother dendrite. Then, WRC recruits the actin elongation factor UNC-34/Ena/VASP to initiate growth of a new dendritic branch from the swelling, with the help of the actin-binding protein UNC-115/abLIM. Extension of existing dendrites also proceeds via swelling formation at the dendrite tip followed by UNC-34-mediated outgrowth. Following dendrite initiation and extension, the stabilization of branches by guidance receptors further recruits WRC, resulting in an iterative process to build a complex dendritic arbor.

The skeletal muscle ryanodine receptor (RyR1) proteins are intracellular calcium (Ca2+) release channels on the membrane of the sarcoplasmic reticulum (SR) and required for skeletal muscle excitation-contraction coupling. Homer (Vesl) is a family of scaffolding proteins that modulate target proteins including RyRs (ryanodine receptors), mGluRs (group 1 metabotropic glutamate receptors) and IP3Rs (inositol-1,4,5-trisphosphate receptors) through a conserved EVH1 (Ena/VASP homology 1) domain. Here, we examined the interaction between Homer1 EVH1 domain and RyR1 by co-immunoprecipitation, continuous sucrose density-gradient centrifugation, and bio-layer interferometry binding assay at different Ca2+ concentrations. Our results show that there exists a high-affinity binding between the Homer1 EVH1 domain and RyR1, especially at 1 mM of Ca2+. Based on our data and the known structures of Homer1 EVH1 domain and RyR1, we found two consensus proline-rich sequences in the structure of RyR1, PPHHF and FLPPP, and proposed two corresponding binding models to show mechanisms of recognition different from those used by other proline-rich motifs. The side proline residues of two proline-rich motifs from RyR1 are away from the hydrophobic surface of Homer1 EVH1, rather than buried in this hydrophobic surface. Our results provide evidence that Homer1 regulates RyR1 by direct interaction.

The human proteome is replete with short linear motifs (SLiMs) of 4-6 residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to systematically examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of ENAH, an actin regulator that is upregulated in invasive cancers, we screened 36-residue proteome-derived peptides for binding. We discovered a pocket on the ENAH EVH1 domain that diverged from its orthologs to recognize extended SLiMs, and we found that proteins with two EVH1-binding SLiMs can wrap around a single domain. We also found that the ciliary protein PCARE uses an extended 23-residue region to obtain higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of broader proteomic context on motif-mediated interactions, revealing diverse mechanisms of contextual control over EVH1 interactions and establishing that SLiMs can’t be fully understood outside of their native context.

Peer reviewed: True, Protein phosphatase 4 (PP4) is an evolutionarily conserved and essential Ser/Thr phosphatase that regulates cell division, development and DNA repair in eukaryotes. The major form of PP4, present from yeast to human, is the PP4c-R2-R3 heterotrimeric complex. The R3 subunit is responsible for substrate-recognition via its EVH1 domain. In typical EVH1 domains, conserved phenylalanine, tyrosine and tryptophan residues form the specific recognition site for their target's proline-rich sequences. Here, we identify novel binding partners of the EVH1 domain of the Drosophila R3 subunit, Falafel, and demonstrate that instead of binding to proline-rich sequences this EVH1 variant specifically recognizes atypical ligands, namely the FxxP and MxPP short linear consensus motifs. This interaction is dependent on an exclusively conserved leucine that replaces the phenylalanine invariant of all canonical EVH1 domains. We propose that the EVH1 domain of PP4 represents a new class of the EVH1 family that can accommodate low proline content sequences, such as the FxxP motif. Finally, our data implicate the conserved Smk-1 domain of Falafel in target-binding. These findings greatly enhance our understanding of the substrate-recognition mechanisms and function of PP4.

Significance Protein–protein interactions mediated by proline-rich motifs are involved in regulation of many important signaling cascades. These motifs belong to the most abundant recognition motifs in the eukaryotic genome and preferentially adopt a left-handed polyproline helix II, a secondary structure element that has been notoriously difficult to mimic with small molecules. Here, we present a structure-guided design effort yielding a toolkit of chemical entities that enables rational construction of selective small molecule inhibitors for these protein domains. We succeeded in developing an inhibitor for the Ena/VASP protein family that is active in vivo and reduces extravasation of invasive breast cancer cells in a zebrafish model., Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor (Kd=120 nM,MW=734 Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24–45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26–37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells., Author summary The delta-retrovirus Human T-cell leukemia virus type 1 encodes the accessory protein p8, which is generated by proteolytic cleavage from p12. Earlier work has shown that p8 enhances the formation of cellular conduits between T-cells, is transferred through these conduits to target T-cells and increases HTLV-1 transmission. It was suggested that p8 dampens T-cell responses in target T-cells, thus facilitating HTLV-1 infection. Our work sheds light on the mechanism of p8 transfer to target T-cells. We show that vasodilator-stimulated phosphoprotein (VASP), a novel interaction partner of p8, contributes to transfer of p8 to target T-cells. Mechanistically, VASP is crucial for recruitment of p8 to the cell surface. Since VASP is known to promote elongation of actin filaments by preventing them from capping, interactions of p8 with VASP are an elegant strategy to exploit the host cell machinery for being transported to the cell surface, and as a consequence, to other cells. Given that VASP is also important for cell-to-cell transfer of the HTLV-1 Gag protein, our work proposes that VASP is a new cellular target to counteract HTLV-1 cell-to-cell transmission.

In this issue of Structure, Li et al. (2018) describe a crystallographic view of the Drebrin-Homer interaction, mediated by the Homer binding motif of Drebrin and EVH1 domain of Homer. This interaction enables cross-linking of monomeric Drebrin by tetrameric Homer, which is required for efficient F-actin bundling.

Human Pre-mRNA Processing factor 19 (hPRPF19) is an important component in human spliceosome machinery. hPRPF19 contains a WD40 repeats domain at its C-terminus, which is also conserved in yeast. Here we determined the crystal structure of the C-terminal WD40 repeat domain of hPRPF19 by X-ray crystallography. Our structural analysis revealed some significantly different structure features between the human and yeast Prp19 WD40 repeat domain. However, there are also conserved clusters of residues at the bottom surface of the fourth and the fifth WD40 repeats, which provides the important implication for the conserved Prp19 proteins in both human and yeast.

• TRPV6 is one of the most Ca 2+ selective Trp ion channels. It is mainly expressed in Ca 2+ -transporting epithelia e.g. epididymis and intestine, where it is assumed to play an important role in Ca 2+ (re)absorption.

The 106-residue protein Q4DY78 (UniProt accession number) from Trypanosoma cruzi is highly conserved in the related kinetoplastid pathogens Trypanosoma brucei and Leishmania major. Given the essentiality of its orthologue in T. brucei, the high sequence conservation with other trypanosomatid proteins, and the low sequence similarity with mammalian proteins, Q4DY78 is an attractive protein for structural characterization. Here, we solved the structure of Q4DY78 by solution NMR and evaluated its backbone dynamics. Q4DY78 is composed of five α -helices and a small, two-stranded antiparallel β-sheet. The backbone RMSD is 0.22 ± 0.05 A for the representative ensemble of the 20 lowest-energy structures. Q4DY78 is overall rigid, except for N-terminal residues (V8 to I10), residues at loop 4 (K57 to G65) and residues at the C-terminus (F89 to F112). Q4DY78 has a short motif FPCAP that could potentially mediate interactions with the host cytoskeleton via interaction with EVH1 (Drosophila Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) homology 1) domains. Albeit Q4DY78 lacks calcium-binding motifs, its fold resembles that of eukaryotic calcium-binding proteins such as calcitracin, calmodulin, and polcacin Bet V4. We characterized this novel protein with a calcium binding fold without the capacity to bind calcium.

Vasodilator-stimulated phosphoprotein (VASP) is a processive actin polymerase with roles in the control of cell shape and cell migration. Through interaction with the cytoskeletal adaptor protein Zyxin, VASP can localize to damaged stress fibers where it serves to repair and reinforce these structures. VASP localization is mediated by its N-terminal Ena/VASP homology (EVH1) domain, which binds to the (W/F)PxφP motif (most commonly occurring as FPPPP) found in cytoskeletal proteins such as vinculin, lamellipodin, and Zyxin. Sequentially close clusters of four or five of these motifs frequently occur, as in the proline rich region of Zyxin with four such motifs. This suggests that tetrameric VASP might bind very tightly to Zyxin through avidity, with all four EVH1 domains binding to a single Zyxin molecule. Here, quantitative nuclear magnetic resonance titration analysis reveals a dominant bivalent 1:1 (Zyxin:EVH1) interaction between the Zyxin proline rich region and the VASP EVH1 domain that utilizes the EVH1 canonical binding site and a novel secondary binding site on the opposite face of the EVH1 domain. We further show that binding to the secondary binding site is specifically inhibited by mutation of VASP EVH1 domain residue Y39 to E, which mimics Abl-induced phosphorylation of Y39. On the basis of these findings, we propose a model in which phosphorylation of Y39 acts as a stoichiometry switch that governs binding partner selection by the constitutive VASP tetramer. These results have broader implications for other multivalent VASP EVH1 domain binding partners and for furthering our understanding of the role of Y39 phosphorylation in regulating VASP localization and cellular function.

Neurofibromin and Sprouty-related EVH1 domain-containing protein 1 (Spred1) both act as negative regulators of the mitogen-activated protein kinase pathway and are associated with the rare diseases Neurofibromatosis type 1 and Legius syndrome, respectively. Spred1 recruits the major GTPase activating protein (GAP) neurofibromin from the cytosol to the membrane in order to inactivate the small G protein Ras. These functions are dependent on the N-terminal EVH1 domain and the C-terminal Sprouty domain of Spred1 whereas the former specifically recognizes the GAP related domain of neurofibromin and the latter is responsible for membrane targeting. Within the GAP domain, Spred1 binding depends on the GAPex portion which is dispensable for Ras inactivation. In a first step towards the characterization of the Neurofibromin Spred1 interface in solution we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of the Spred1 derived EVH1 domain. Our chemical shift data analysis indicate seven consecutive β-strands followed by a C-terminal α-helix which is in agreement with the previously reported crystal structure of Spred1(EVH1). Our data provide a framework for further analysis of the function of patient-derived mutations associated with rare diseases.

The Shank family comprises three large multi-domain proteins playing central roles as protein scaffolds in the neuronal postsynaptic density. The Shank proteins are closely linked to neuropsychiatric diseases, such as autism spectrum disorders. One characteristic domain in the Shank family is the SH3 domain, assumed to play a role in protein-protein interactions; however, no specific ligand binding to any Shank SH3 domain has been described. We solved the crystal structure of the SH3 domain from Shank3 at sub-atomic resolution. While the structure presents the canonical SH3 domain fold, the binding site for proline-rich peptides is not conserved. In line with this, no binding of Pro-rich sequences by the Shank3 SH3 domain was observed. Sequence comparisons indicate that all Shank isoforms have similarly lost the classical Pro-rich peptide binding site from the SH3 domain. Whether the corresponding site in the Shank SH3 domains has evolved to bind a non-poly-Pro target sequence is currently not known. Our work provides an intriguing example of the evolution of a well-characterized protein-protein interaction domain within the context of multi-domain protein scaffolds, allowing the conservation of structural features, but losing canonical functional sites. The data are further discussed in light of known mutations in the SH3 domain or its vicinity in the different Shank isoforms.

Slingshot-1 (SSH1) is a protein phosphatase that specifically dephosphorylates and activates cofilin, an F-actin-severing protein. SSH1 binds to and co-localizes with F-actin, and the cofilin-phosphatase activity of SSH1 is markedly increased by binding to F-actin. In this study, we performed a secondary structure analysis of SSH1, which predicted the existence of a pleckstrin homology (PH)-like domain in the N-terminal region of SSH1. SSH1 also contains a DEK-C domain in the N-terminal region. The N-terminal fragment of SSH1 bound to and co-localized with F-actin, but mutation at Arg-96 or a Leu-His-Lys (LHK) motif in the PH-like domain reduced this activity. Furthermore, mutation at Arg-96 abrogated the cofilin-phosphatase activity of SSH1 in the presence of F-actin. These results suggest that the N-terminal PH-like domain plays a critical role in F-actin binding and F-actin-mediated activation of the cofilin-phosphatase activity of SSH1.

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance system that maintains the integrity of transcripts by removing aberrant mRNAs harbouring premature termination codons (PTCs). In the absence of NMD, truncated proteins with dominant negative or potentially deleterious gain of function activity may be expressed from these erroneous mRNAs. In addition to its function as a quality control for mRNAs, NMD is involved in the regulation of 3 – 10% of the Saccharomyces cerevisiae, Drosophilia melanogaster, and Homo sapiens transcriptomes. PTC recognition in NMD requires the cross talk between terminating ribosome stalled at stop codon and downstream cis-acting signal that is not conserved across species. In S. cerevisiae and D. melanogaster, the faux 3’ untranslated region (UTR) model has been proposed to be the main mechanism in recognising PTC. In mammals, PTC recognition is mediated through the interaction of the SMG1, Upf1, eukaryotic Release factor 1 and 3 (SURF) complex on the terminating ribosome at a PTC and downstream exon junction complex (EJC) during the pioneer round of translation. Upon recognition of PTC, an evolutionarily conserved surveillance complex consisting of the Upf1, Upf2 and Upf3 proteins is assembled. In mammals, the assembly of the surveillance complex also activates a phosphoinositide-3-kinase related protein kinase, SMG1, which will phosphorylate the Upf1 protein. Till date, three mechanisms of how phosphorylated Upf1 protein leads to decay of mRNAs have been proposed. First, the phosphorylated Upf1 protein recruits the SMG7 protein and C terminal of SMG7 protein targets the associated mRNA for decay through an undetermined mechanism. Second, the phosphorylated Upf1 protein recruitshDcp1a or Dcp2 proteins to decap mRNA for its subsequent 5’-3’ decay. Third, the hyperphosphorylated Upf1 protein associates with the nuclear receptor coregulatory protein 2 (PNRC2), which targets the mRNA to the P body through the interaction between the PNRC2 and hDcp1a proteins. Subsequently, decapping and 5’ - 3’ decay of mRNA occurs in the P body. As the three mechanisms are inferred from the results of coimmunoprecipitation or two-hybrid interactions, no direct interaction between the hyperphosphorylated Upf1 protein and decapping enzymes has been demonstrated till date. Here we present the crystal structure of hDcp1a protein in complex with the PNRC2 peptide. The proline-rich region of the PNRC2 peptide is bound to the EVH1 domain of the hDcp1a protein while isothermal titration calorimetry study demonstrates that NR-box of the PNRC2 protein mediates the direct interaction with hyperphosphorylated Upf1 protein. The mode of the PNRC2 protein interaction with the hDcp1a protein is distinct from those observed in other classes of EVH1/proline-rich ligands interactions. Additionally, PNRC2 mutagenesis study performed by our collaborator showed that disruption of the interaction of the PNRC2 protein with the hDcp1a protein abolishes its P-body localization and ability to promote mRNA degradation when tethered to mRNAs. Furthermore, the PNRC2 protein acts in synergy with the hDcp1a protein to stimulate the decapping activity of the Dcp2 protein by bridging the interaction between the hDcp1a and Dcp2 proteins. The formation of a novel PNRC2-hDcp1a-Dcp2 decapping complex suggests that the PNRC2 protein is a decapping coactivator in addition to its adaptor role in NMD. DOCTOR OF PHILOSOPHY (SBS)

Motivation Polyproline II (PPII) is a common conformation, comparable to α-helix and β-sheet. PPII, recently termed with a more generic name—κ-helix, adopts a left-handed structure with 3-fold rotational symmetry. Lately, a new type of binding mechanism—the helical lock and key model was introduced in SH3-domain complexes, where the interaction is characterized by a sliding helical pattern. However, whether this binding mechanism is unique only to SH3 domains is unreported. Results Here, we show that the helical binding pattern is a universal feature of the κ-helix conformation, present within all the major target families—SH3, WW, profilin, MHC-II, EVH1 and GYF domains. Based on a geometric analysis of 255 experimentally solved structures, we found that they are characterized by a distinctive rotational angle along the helical axis. Furthermore, we found that the range of helical pitch varies between different protein domains or peptide orientations and that the interaction is also represented by a rotational displacement mimicking helical motion. The discovery of rotational interactions as a mechanism, reveals a new dimension in the realm of protein–protein interactions, which introduces a new layer of information encoded by the helical conformation. Due to the extensive involvement of the conformation in functional interactions, we anticipate our model to expand the current molecular understanding of the relationship between protein structure and function. Availability and implementation We have implemented the proposed methods in an R package freely available at https://github.com/Grantlab/bio3d. Supplementary information Supplementary data are available at Bioinformatics online.

Neurofibromatosis type I (NF1) and Legius syndrome are rare inherited disorders that share diagnostic symptoms including dermal abnormalities like axillary and inguinal freckling and cafe au lait spots. In addition, patients suffering from NF1 have a demanding risk for the development of severe tumors of the peripheral and central nervous system among other NF1-specific symptoms. NF1 and Legius syndrome are caused by alterations in the NF1 and SPRED1 genes encoding the Ras inhibitors neurofibromin and Spred1 (sprouty related EVH1 domain-containing protein), respectively. Neurofibromin functions as a Ras-specific GTPase-activating protein (Ras-GAP), and Spred1 enhances Ras inactivation by recruiting neurofibromin from the cytosol to membrane-anchored Ras. In a previous study, we mapped the Spred binding site to the GAP-related domain of neurofibromin (NF1-GAP) and identified the GAPex subdomain as critical for Spred1 binding. Here, we characterize the binding site of these proteins in more detail focusing on a mutant Spred1 variant carrying a pathogenic missense mutation (threonine 102 to arginine). Introduction of this mutation, which locates at the N-terminal EVH1 domain of Spred1, weakens the interaction with neurofibromin by about 3 orders of magnitude without perturbing the protein fold, and the binding site of NF1-GAP on the mutant Spred1(EVH1) variant can be identified by NMR spectroscopy. Taken together, our data provide structural insight into the interaction of Spred1 and neurofibromin and characterize the structural or functional consequence of selected patient-derived mutations associated with Legius syndrome.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

The DCP1-DCP2 complex can regulate the animal antiviral immunity by the decapping of retrovirus RNAs and the suppression of RNAi pathway. However, the influence of DCP1-DCP2 complex on DNA virus infection and the regulation of DCP1-DCP2 complex by microRNAs (miRNAs) remain unclear. In this study, we investigated the role of miRNA-regulated DCP1-DCP2 complex in DNA virus infection. Our results suggested that the DCP1-DCP2 complex played a positive role in the infection of white spot syndrome virus (WSSV), a DNA virus of shrimp. The N-terminal regulatory domain of DCP2 was interacted with the EVH1 domain of DCP1, forming the DCP1-DCP2 complex. Furthermore, a host shrimp miRNA (miR-87) inhibited WSSV infection by targeting the host DCP2 gene and a viral miRNA (WSSV-miR-N46) took a negative effect on WSSV replication by targeting the host DCP1 gene. Therefore, our study provided novel insights into the underlying mechanism of DCP1-DCP2 complex and its regulation by miRNAs in virus-host interactions.The DCP1-DCP2 complex can regulate the animal antiviral immunity by the decapping of retrovirus RNAs and the suppression of RNAi pathway. In the present study, the findings indicated that the silencing of the DCP1-DCP2 complex inhibited the infection of WSSV, a DNA virus of shrimp, suggesting that the DCP1-DCP2 complex facilitated DNA virus infection. Due to the suppressive role of the DCP1-DCP2 complex in RNAi pathway against virus infection, the DCP1-DCP2 complex could promote WSSV infection in shrimp. In this context, our study contributed a novel aspect of the DCP1-DCP2 complex in virus-host interactions. Our study revealed that the host and viral miRNAs could regulate the DCP1-DCP2 complex to affect virus infection. Therefore, our study provided novel insights into the miRNA-mediated regulation of DCP1-DCP2 complex took great effects on RNAi immunity of invertebrates against virus infection.

The guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2) is identified as a member of the Dbl family of GEFs. It regulates RhoA-dependent cell signaling pathways and plays important roles in biological processes. GEF-H1 contains an N-terminal zinc finger domain, a Dbl-homologous (DH) domain followed by a Pleckstrin homology (PH) domain, and a C-terminal domain. The specific roles of its PH domain are poorly understood. Here we report the crystal structure of human GEF-H1 PH domain to 2.45 Å resolution. A conserved surface is formed by β8, β9, β10, and it may mediate protein-protein interactions. Although the folding resembles other PH domains that have defined structures, superposition of different PH domains clearly shows that the loop between β6/β7 and the loop between β3/β4 are so close that they will prevent its binding with phosphoinositide due to steric hindrance, and this has been proved by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Our studies provide a structural framework for further work on the function of GEF-H1.

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple cafe-au-lait macules, axillary freckling, learning disabilities and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the RAS-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids (aa) and the C-terminal 20 aa of the GTPase-Activating Protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120GAP. Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome. ispartof: Journal of Biological Chemistry vol:291 issue:7 pages:3124-34 ispartof: location:United States status: published

Shigella spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. Shigella utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to Shigella flexneri spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting S. flexneri spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in Shigella pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells.

SUMMARY Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions., Graphical Abstract, In Brief Yan et al. describe the structure of neurofibromin (GAP-related domain) in complex with SPRED1(EVH1) and KRAS and suggest a mechanism by which this interaction is regulated during normal cell signaling and in cells with activated receptor tyrosine kinase oncogenes. The neurofibromin-SPRED1 interaction interface provides a rationale for mutations observed in Legius syndrome.

The protein hSos1 is a Ras guanine nucleotide exchange factor. In the present study, we investigated the function of the amino-terminal region of the hSos1 protein, corresponding to the first 600 residues, which includes the Dbl and pleckstrin homology (DH and PH) domains. We demonstrated, using a series of truncated mutants, that this region is absolutely necessary for hSos1 activity. Our results suggest that the first 200 residues (upstream of DH domain), which we called the HF motif on the basis of their homology with histone H2A, may exert negative control over the functional activity of the whole hSos1 protein. In vitro binding analysis showed that the HF motif is able to interact specifically with the PH domain of hSos1. The amino-terminal region of hSos1 may be associated in vivo with an expressed HF motif. These findings document the existence of the HF motif located upstream of the DH domain in the hSos1 protein. This motif may be responsible for the negative control of hSos1, probably by intramolecular binding with the PH domain.

Summary Drebrin is an actin bundling protein that plays critical roles in synaptic spine development and plasticity. Homer, one of the most abundant scaffolding proteins in postsynaptic density, interacts with Drebrin's C-terminal PPXXF motifs using its Ena/VASP homology 1 (EVH1) domain. However, the molecular mechanism and biological function of this interaction remain unclear. Here we show that Homer specifically binds to the first but not the second PPXXF motif in Drebrin. The crystal structure of Drebrin-Homer binding motif 1 in complex with Homer EVH1 reveals a consensus Homer EVH1 binding motif. Homer tetramer promotes actin bundling activity of Drebrin in vitro and stimulates Drebrin-induced filopodia formation and elongation in cells. We further show that monomeric Homer1a antagonizes Homer1b in promoting Drebrin-stimulated actin bundling. Our study suggests a potential regulatory role of Homer1 in modulating excitatory synaptic spine homeostatic scaling via binding to Drebrin.

Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X) n L ( n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X) n L motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X) n L-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X) n L motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.

With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.

Amyloid-β protein precursor (AβPP) metabolism and the accumulation of its derivative amyloid-β (Aβ) peptide in senile plaques have been considered key players in the development of Alzheimer's disease (AD). However, the mechanisms underlying the generation and the deposition of Aβ are not clear but emphasis has been given in the role of AβPP protein interactions that regulate its processing and offer a means to manipulate Aβ production. We have previously shown that AβPP interacts with members of the Homer protein family, which leads to inhibition of Aβ generation. Herein, we studied the structural parameters of AβPP/Homer3 interaction by analyzing the sequences and domains that play a role in the formation of the complex. We found that the cytoplasmic tail of AβPP is necessary for the interaction. Regarding Homer3, we report that both the EVH1 protein interacting domain and the polymerization coiled coil domain are essential for the complex assembly. Importantly, phosphorylation of Homers at certain serine residues seems to enhance the interaction with AβPP, possibly underlying our recent work suggesting that calcium signaling also regulates the interaction. Our results show that the regulation of AβPP/Homer3 interaction might be critical in the context of Alzheimer's disease pathology as a novel target for regulating AβPP function and metabolism.

A large family of G protein-coupled receptors (GPCRs) involved in cell adhesion has a characteristic autoproteolysis motif of HLT/S known as the GPCR proteolysis site (GPS). GPS is also shared by polycystic kidney disease proteins and it precedes the first transmembrane segment in both families. Recent structural studies have elucidated the GPS to be part of a larger domain named GPCR autoproteolysis inducing (GAIN) domain. Here we demonstrate the remote homology relationships of GAIN domain to ZU5 domain and Nucleoporin98 (Nup98) C-terminal domain by structural and sequence analysis. Sequence homology searches were performed to extend ZU5-like domains to bacteria and archaea, as well as new eukaryotic families. We found that the consecutive ZU5-UPA-death domain domain organization is commonly used in human cytoplasmic proteins with ZU5 domains, including CARD8 (caspase recruitment domain-containing protein 8) and NLRP1 (NACHT, LRR and PYD domain-containing protein 1) from the FIIND (Function to Find) family. Another divergent family of extracellular ZU5-like domains was identified in cartilage intermediate layer proteins and FAM171 proteins. Current diverse families of GAIN domain subdomain B, ZU5 and Nup98 C-terminal domain likely evolved from an ancient autoproteolytic domain with an HFS motif. The autoproteolytic site was kept intact in Nup98, p53-induced protein with a death domain and UNC5C-like, deteriorated in many ZU5 domains and changed in GAIN and FIIND. Deletion of the strand after the cleavage site was observed in zonula occluden-1 and some Nup98 homologs. These findings link several autoproteolytic domains, extend our understanding of GAIN domain origination in adhesion GPCRs and provide insights into the evolution of an ancient autoproteolytic domain.

The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the SLC4 family. A novel conserved Zn2+-binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn2+ dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn2+ site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

The suppressor domain of inositol 1,4,5 trisphosphate receptor (IP3R) has critical roles in regulating the calcium channel by interacting with many binding partners. The residue 49–53 (PPKKF) of the suppressor domain was suggested to be a canonical Homer EVH1 domain binding site and is also the first a part of calmodulin (CaM) binding site. As CaM-binding of the suppressor domain has been shown to involve large-scale conformational changes, we studied the binding characteristics of the Homer EVH1-suppressor domain with NMR spectroscopy and biochemical pull-down assays for mutants. Our data show that the suppressor domain employs the PPKKF motif in a similar but subtly different way compared to previously characterized interactions, and that the suppressor domain does not undergo large-scale conformational changes. Chemical shift assignments of the Homer3 EVH1 domain found that a new set of residues, located at the opposite side of the previously reported binding site, is also involved in binding, which was confirmed by mutant binding assays. Further analysis suggests that F40 in the new binding sites may have a critical role as a conformational lock-switch in Homer-target binding. The proposed mechanism is implicated in the signaling network involving calcium channels.

Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP–IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-ε activation. We show that the monomeric VASP EVH1 domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 μM). We further show that this motif adopts distinct cis and trans isomers for the Trp168–Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the binding reaction, showing diffusion-limited binding to the trans isomer followed by slow, isomerization-dependent binding. We further demonstrate that the peptidyl-prolyl isomerase cyclophilin A (CypA) catalyzes isomerization of the Trp168–Pro169 peptide bond and accelerates binding of the IRAK1-UD to the VASP EVH1 domain. We propose that binding of IRAK1 to tetrameric VASP is regulated by avidity through the assembly of IRAK1 onto receptor-anchored signaling complexes and that an isomerase such as CypA may modulate IRAK1 signaling in vivo. These studies demonstrate a direct interaction between IRAK1 and VASP and suggest a potential mechanism for how this interaction might be regulated by both assembly of IRAK1 onto an activated signaling complex and PPIase enzymes.

Scml2 is a member of the Polycomb group of proteins involved in epigenetic gene silencing. Human Scml2 is a part of a multisubunit protein complex, PRC1 (Polycomb repressive complex 1), which is responsible for maintenance of gene repression, prevention of chromatin remodeling, preservation of the "stemness" of the cell, and cell differentiation. Although the majority of PRC1 subunits have been recently characterized, the structure of Scml2 and its role in PRC1-mediated gene silencing remain unknown. In this work a conserved protein domain within human Scml2 has been identified, and its structure was determined by solution NMR spectroscopy. This module was named Scm-like embedded domain, or SLED. Evolutionarily, the SLED domain emerges in the first multicellular organisms, consistent with the role of Scml2 in cell differentiation. Furthermore, it is exclusively found within the Scm-like family of proteins, often accompanied by malignant brain tumor domain (MBT) and sterile α motif (SAM) domains. The domain adopts a novel α/β fold with no structural analogues found in the Protein Data Bank (PDB). The ability of the SLED to bind double-stranded DNA was also examined, and the isolated domain was shown to interact with DNA in a sequence-specific manner. Because PRC1 complexes localize to the promoters of a specific subset of developmental genes in vivo, the SLED domain of Scml2 may provide an important link connecting the PRC1 complexes to their target genes.

TAX1BP1 is a highly conserved, pleiotropic protein that plays many essential functions in human cells, including negative regulation of inflammatory and antimicrobial responses mediated by NF-κB and IRF3 signaling, inhibition of apoptosis, transcriptional coactivation and autophagyetc.TAX1BP1 contains a SKICH domain at the N-terminus, three coiled-coil domains in the middle and two ubiquitin-binding zinc-finger motifs at the C-terminus. The SKICH domain and the linker sequence between the SKICH domain and the coiled-coil region mediate interaction with ubiquitin-like proteins of the LC3/GABARAP family, which are autophagosome markers. For structure determination of the SKICH domain of TAX1BP1, a protein construct (amino acids 15–148) corresponding to the SKICH domain plus the linker region was expressed, purified and crystallized. A native diffraction data set has been collected to 1.9 Å resolution. A molecular-replacement solution has been found by using the structure of the SKICH domain of NDP52, a paralog of TAX1BP1.

In this paper, we describe the structure of a N-terminal domain motif in nuclear-localized FKBP251-73, a member of the FKBP family, together with the structure of a sequence-related subdomain of the E3 ubiquitin ligase HectD1 that we show belongs to the same fold. This motif adopts a compact 5-helix bundle which we name the Basic Tilted Helix Bundle (BTHB) domain. A positively charged surface patch, structurally centered around the tilted helix H4, is present in both FKBP25 and HectD1 and is conserved in both proteins, suggesting a conserved functional role. We provide detailed comparative analysis of the structures of the two proteins and their sequence similarities, and analysis of the interaction of the proposed FKBP25 binding protein YY1. We suggest that the basic motif in BTHB is involved in the observed DNA binding of FKBP25, and that the function of this domain can be affected by regulatory YY1 binding and/or interactions with adjacent domains.

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.

We have shown previously that poliovirus infection disrupts cytoplasmic P-bodies in infected mammalian cells. During the infectious cycle, poliovirus causes the directed cleavage of Dcp1a and Pan3, coincident with the dispersion of P-bodies. We now show that expression of Dcp1a prior to infection, surprisingly, restricts poliovirus infection. This inhibition of infection was independent of P-body formation because expression of GFP-Dcp1a mutants that cannot enter P-bodies restricted poliovirus infection similar to wild-type GFP-Dcp1a. Expression of wild-type or mutant GFP-Dcp1a induced phosphorylation of eIF2α through the eIF2α kinase protein kinase R (PKR). Activation of PKR required the amino-terminal EVH1 domain of Dcp1a. This PKR-induced translational inhibition appears to be specific to Dcp1a because the expression of other P-body components, Pan2, Pan3, Ccr4, or Caf1, did not result in the inhibition of poliovirus gene expression or induce eIF2α phosphorylation. The translation blockade induced by Dcp1a expression suggests novel signaling linking RNA degradation/decapping and regulation of translation.

The Homer family of proteins consists of three adaptor proteins, Homer1, Homer2 and Homer3, each with various isoforms. Homer1 family presents an EVH1 domain, a coiled coil domain and two leucine zipper domains. Homer proteins regulate a number of Ca2+-handling proteins, including transient receptor potential channels and other Ca2+-permeable channels, ionotropic and metabotropic glutamate receptors, shank scaffolding proteins or endoplasmic reticulum Ca2+ release channels. This review article focuses on the association of Homer 1 proteins with Ca2+-handling proteins and their role on intracellular Ca2+-homeostasis.

The casein kinase 2 interacting protein-1 (CKIP-1) is involved in many cellular functions, including apoptosis, signalling pathways, cell growth, cytoskeleton and bone formation. Its N-terminal pleckstrin homology (PH) domain is thought to play an important role in membrane localization and controls shuttling of CKIP-1 between the plasma membrane and nucleus. In this study, the human CKIP-1 PH domain was purified but problems were encountered with nucleic acid contamination. An S84D/S86D/S88D triple mutant designed to abolish nucleic acid binding was purified and successfully crystallized. Single crystals diffracted to 1.7 A resolution and belonged to space group P43212 with unit-cell parameters a = 53.0, b = 53.0, c = 113.8 A, α = β = γ = 90.0°.

Intersectin-2L (ITSN-2L) is a long isoform of ITSN family, which is a multimodule scaffolding protein functioning in membrane-associated molecular trafficking and signal transduction pathways. ITSN-2L possesses a carboxy-terminal extension encoding a Dbl homology domain (DH), a pleckstrin homology domain (PH) and a C2 domain, suggesting that it could act as a guanine nucleotide exchange factor for Rho-like GTPases. But the role of C2 domain is obscure in this process. Here we report the crystal structure of human ITSN-2L C2 domain at 1.56Å resolution. The sequence and structural alignment of ITSN-2L C2 domain with other members of C2 domain protein family indicate its vital cellular roles in membrane trafficking, the generation of lipid-second messengers and activation of GTPases. Moreover, our data show the possible roles of ITSN-2L C2 domain in regulating the activity of Cdc42.