Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs

Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands. Each sequence motif is represented as a position-specific scoring matrix (PSSM) based on results from oriented peptide library and phage display experiments. Predicted domain-motif interactions from Scansite can be sequentially combined, allowing segments of biological pathways to be constructed in silico. The current release of Scansite, version 2.0, includes 62 motifs characterizing the binding and/or substrate specificities of many families of Ser/Thr- or Tyr-kinases, SH2, SH3, PDZ, 14-3-3 and PTB domains, together with signature motifs for PtdIns(3,4,5)P(3)-specific PH domains. Scansite 2.0 contains significant improvements to its original interface, including a number of new generalized user features and significantly enhanced performance. Searches of all SWISS-PROT, TrEMBL, Genpept and Ensembl protein database entries are now possible with run times reduced by approximately 60% when compared with Scansite version 1.0. Scansite 2.0 allows restricted searching of species-specific proteins, as well as isoelectric point and molecular weight sorting to facilitate comparison of predictions with results from two-dimensional gel electrophoresis experiments. Support for user-defined motifs has been increased, allowing easier input of user-defined matrices and permitting user-defined motifs to be combined with pre-compiled Scansite motifs for dual motif searching. In addition, a new series of Sequence Match programs for non-quantitative user-defined motifs has been implemented. Scansite is available via the World Wide Web at http://scansite.mit.edu.     

Bioinformatics--The new home for protein sequence motifs

Protein domains and sequence motifs have been very influentialin the field of molecular biology. These units are the commoncurrency of protein structure and function. The     

Helix-helix interactions and their impact on protein motifs and assemblies

Protein secondary structure elements are arranged in distinct structural motifs such as four-α-helix bundle, 8α/8β TIM-barrel, Rossmann dinucleotide binding fold, assembly of a helical rod. Each structural motif is characterized by a particular type of helix-helix interactions. A unique pattern of contacts is formed by interacting helices of the structural motif. In each type of fold, edges of the helix surface, which participate in the formation of helix-helix contacts with preceding and following helices, differ. This work shows that circular arrangements of the four, eight, and sixteen α-helices, which are found in the four-α-helical motif, TIM-barrel 8α/8β fold, and helical rod of 16.3¯ helices per turn correspondingly, can be associated with the mutual positioning of the edges of the helix surfaces. Edges (i, i+1)−(i+1, i+2) of the helix surface are central for the interhelical contacts in a four-α-helix bundle. Edges (i, i+1)−(i+2, i+3) are involved in the assembly of four-α-helix subunits into helical rod of a tobacco mosaic virus and a three-helix fragment of a Rossmann fold. In 8α/8β TIM-barrel fold, edges (i, i+1)−(i+5, i+6) are involved in the octagon arrangement. Approximation of a cross section of each motif with a polygon (n-gon, n=4, 8, 16) shows that a good correlation exists between polygon interior angles and angles formed by the edges of helix surfaces.     

Characterization of protein hubs by inferring interacting motifs from protein interactions

The characterization of protein interactions is essential for understanding biological systems. While genome-scale methods are available for identifying interacting proteins, they do not pinpoint the interacting motifs (e.g., a domain, sequence segments, a binding site, or a set of residues). Here, we develop and apply a method for delineating the interacting motifs of hub proteins (i.e., highly connected proteins). The method relies on the observation that proteins with common interaction partners tend to interact with these partners through a common interacting motif. The sole input for the method are binary protein interactions; neither sequence nor structure information is needed. The approach is evaluated by comparing the inferred interacting motifs with domain families defined for 368 proteins in the Structural Classification of Proteins (SCOP). The positive predictive value of the method for detecting proteins with common SCOP families is 75% at sensitivity of 10%. Most of the inferred interacting motifs were significantly associated with sequence patterns, which could be responsible for the common interactions. We find that yeast hubs with multiple interacting motifs are more likely to be essential than hubs with one or two interacting motifs, thus rationalizing the previously observed correlation between essentiality and the number of interacting partners of a protein. We also find that yeast hubs with multiple interacting motifs evolve slower than the average protein, contrary to the hubs with one or two interacting motifs. The proposed method will help us discover unknown interacting motifs and provide biological insights about protein hubs and their roles in interaction networks.     

Detecting DNA-binding helix-turn-helix structural motifs using sequence and structure information

In this work, we analyse the potential for using structural knowledge to improve the detection of the DNA-binding helix–turn–helix (HTH) motif from sequence. Starting from a set of DNA-binding protein structures that include a functional HTH motif and have no apparent sequence similarity to each other, two different libraries of hidden Markov models (HMMs) were built. One library included sequence models of whole DNA-binding domains, which incorporate the HTH motif, the second library included shorter models of ‘partial’ domains, representing only the fraction of the domain that corresponds to the functionally relevant HTH motif itself. The libraries were scanned against a dataset of protein sequences, some containing the HTH motifs, others not. HMM predictions were compared with the results obtained from a previously published structure-based method and subsequently combined with it. The combined method proved more effective than either of the single-featured approaches, showing that information carried by motif sequences and motif structures are to some extent complementary and can successfully be used together for the detection of DNA-binding HTHs in proteins of unknown function.     

Protein-protein interactions in the membrane: sequence, structural, and biological motifs

Single-span transmembrane (TM) helices have structural and functional roles well beyond serving as mere anchors to tether water-soluble domains in the vicinity of the membrane. They frequently direct the assembly of protein complexes and mediate signal transduction in ways analogous to small modular domains in water-soluble proteins. This review highlights different sequence and structural motifs that direct TM assembly and discusses their roles in diverse biological processes. We believe that TM interactions are potential therapeutic targets, as evidenced by natural proteins that modulate other TM interactions and recent developments in the design of TM-targeting peptides.     

Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning

DNA sequence is an important determinant of the positioning, stability, and activity of nucleosomes, yet the molecular basis of these effects remains elusive. A "consensus DNA sequence" for nucleosome positioning has not been reported and, while certain DNA sequence preferences or motifs for nucleosome positioning have been discovered, how they function is not known. Here, we report that an unexpected observation concerning the reassembly of nucleosomes during salt gradient dialysis has allowed a breakthrough in our efforts to identify the nucleosomal locations of the DNA sequence motifs that dominate histone-DNA interactions and nucleosome positioning. We conclude that a previous selection experiment for high-affinity, nucleosome-forming DNA sequences exerted selective pressure chiefly on the central stretch of the nucleosomal DNA. This observation implies that algorithms for aligning the selected DNA sequences should seek to optimize the alignment over much less than the full 147 bp of nucleosomal DNA. A new alignment calculation implemented these ideas and successfully aligned 19 of the 41 sequences in a non-redundant database of selected high-affinity, nucleosome-positioning sequences. The resulting alignment reveals strong conservation of several stretches within a central 71 bp of the nucleosomal DNA. The alignment further reveals an inherent palindromic symmetry in the selected DNAs; it makes testable predictions of nucleosome positioning on the aligned sequences and for the creation of new positioning sequences, both of which are upheld experimentally; and it suggests new signals that may be important in translational nucleosome positioning.     

S-Nitrosylation signaling regulates cellular protein interactions

BACKGROUND: S-Nitrosothiols are made by nitric oxide synthases and other metalloproteins. Unlike nitric oxide, S-nitrosothiols are involved in localized, covalent signaling reactions in specific cellular compartments. These reactions are enzymatically regulated. SCOPE: S-Nitrosylation affects interactions involved in virtually every aspect of normal cell biology. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation. MAJOR CONCLUSIONS AND SIGNIFICANCE: S-Nitrosylation is a regulated signaling reaction.     

DNA motifs and sequence periodicities

Genomic DNA sequences contain a wealth of information about the bendability and curvature of the DNA molecule. For example, the well-known 10-11 bp periodicities within genomes can be attributed to supercoiled structures or wrapping around nucleosomes. Such periodic signals have previously been examined mainly based on mono- or dinucleotide correlations. In this study, we generalize this approach and analyze correlation functions of longer motifs such as tetramers or poly(A) sequences. Periodically placed motifs may indicate regular protein binding or curvature signals. We detected various periodic signals e.g. strong 10-11 bp oscillations of periodically placed poly(A), poly(T) or poly(W) stretches. These observations lead to a new view on the intensively studied 10-11 bp periodicities.     

Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells

In MDCK cells, basolateral sorting of most membrane proteins has been shown to depend on distinct cytoplasmic domain determinants. These signals can be divided into those which are related to signals for localization at clathrin-coated pits and those which are unrelated. The LDL receptor bears two tyrosine-containing signals, one of each class, that can independently target receptors from the Golgi complex and from endosomes to the basolateral plasma membrane. We have now investigated the other structural features required for the activity of both determinants. We find that both depend, at least in part, on clusters of 1-3 acidic amino acids located on the COOH-terminal side of each tyrosine. While single residues adjacent to each tyrosine were also found to be critical, the two signals differed in that only the coated pit-unrelated signal could tolerate a phenylalanine in place of its tyrosine residue. We also found that the structural requirements for basolateral targeting of the "coated pit-related" signal were distinct from those required for rapid endocytosis. Apart from sharing a common tyrosine residue, no feature of the NPXY motif for coated pit localization was required for basolateral targeting. We also investigated basolateral targeting of the mouse macrophage Fc receptor (FcRII-B2) which contains a tyrosine-independent coated pit localization signal. Basolateral transport and endocytosis were found to depend on a common dileucine-type motif. Thus, basolateral targeting determinants, like coated pit domains, can contain either tyrosine- or di-leucine-containing signals. The amino acids in the vicinity of these motifs determine whether they function as determinants for endocytosis, basolateral targeting, or both.     

3MOTIF: visualizing conserved protein sequence motifs in the protein structure database

SUMMARY: 3MOTIF is a web application that visually maps conserved sequence motifs onto three-dimensional protein structures in the Protein Data Bank (PDB; Berman et al., Nucleic Acids Res., 28, 235-242, 2000). Important properties of motifs such as conservation strength and solvent accessible surface area at each position are visually represented on the structure using a variety of color shading schemes. Users can manipulate the displayed motifs using the freely available Chime plugin. AVAILABILITY: http://motif.stanford.edu/3motif/     

MultiLoc: prediction of protein subcellular localization using N-terminal targeting sequences, sequence motifs and amino acid composition

MOTIVATION: Functional annotation of unknown proteins is a major goal in proteomics. A key annotation is the prediction of a protein's subcellular localization. Numerous prediction techniques have been developed, typically focusing on a single underlying biological aspect or predicting a subset of all possible localizations. An important step is taken towards emulating the protein sorting process by capturing and bringing together biologically relevant information, and addressing the clear need to improve prediction accuracy and localization coverage. RESULTS: Here we present a novel SVM-based approach for predicting subcellular localization, which integrates N-terminal targeting sequences, amino acid composition and protein sequence motifs. We show how this approach improves the prediction based on N-terminal targeting sequences, by comparing our method TargetLoc against existing methods. Furthermore, MultiLoc performs considerably better than comparable methods predicting all major eukaryotic subcellular localizations, and shows better or comparable results to methods that are specialized on fewer localizations or for one organism. AVAILABILITY: http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc/     

Protein sorting and membrane-mediated interactions

Sorting of membrane proteins is of vital importance for living cells. Indeed, roughly one-third of a eukaryotic cell’s proteome consists of peripheral and transmembrane proteins. These need to be properly distributed and dynamically maintained at distinct locations in the compartmentalized cell, and one may wonder how proteins determine where, when, and how to travel to reach a specific organelle. While specific binary interactions between proteins have been invoked in explaining the trafficking and sorting processes, a more active role of lipids in this context has become visible in recent years. In particular, membrane-mediated interactions have been suggested to serve as a robust physicochemical mechanism to facilitate protein sorting. Here, we will review some recent insights into these aspects.     

Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.     

Focal adhesion kinase: protein interactions and cellular functions

Integrin-mediated cell adhesion to extracellular matrix (ECM) plays important roles in a variety of biological processes. Recent studies suggested that integrins mediate signal transduction across the plasma membrane via activating several intracellular signaling pathways. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been shown to be a major mediator of integrin signal transduction pathways. Upon activation by integrins, FAK undergoes autophosphorylation as well as associations with several other intracellular signaling molecules. These interactions in the signaling pathways have been shown to regulation a variety of cellular functions such as cell spreading, migration, cell proliferation, apoptosis and cell survival. Recent progress in the understanding of FAK interactions with other proteins in the regulation of these cellular functions will be discussed in this review.     

Differential protein–protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways

Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1‐ and LRRK2‐specific cellular processes by identifying their distinct interacting proteins. A protein microarray‐based interaction screen was performed with recombinant 3xFlag‐LRRK1 and 3xFlag‐LRRK2 and, in parallel, co‐immunoprecipitation followed by mass spectrometry was performed from SH‐SY5Y neuroblastoma cell lines stably expressing 3xFlag‐LRRK1 or 3xFlag‐LRRK2. We identified a set of LRRK1‐ and LRRK2‐specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF‐R) as a LRRK1‐specific interactor, while 14‐3‐3 proteins were LRRK2‐specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14‐3‐3 consensus binding motifs. To assess the functional relevance of these interactions, SH‐SY5Y‐LRRK1 and ‐LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14‐3‐3 binding, or with EGF, an EGF‐R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins.