Yeast Protein Interactome topology provides framework for coordinated-functionality

The architecture of the network of protein–protein physical interactions in Saccharomyces cerevisiae is exposed through the combination of two complementary theoretical network measures, betweenness centrality and ‘Q-modularity’. The yeast interactome is characterized by well-defined topological modules connected via a small number of inter-module protein interactions. Should such topological inter-module connections turn out to constitute a form of functional coordination between the modules, we speculate that this coordination is occurring typically in a pairwise fashion, rather than by way of high-degree hub proteins responsible for coordinating multiple modules. The unique non-hub-centric hierarchical organization of the interactome is not reproduced by gene duplication-and-divergence stochastic growth models that disregard global selective pressures.     

Complementing the Eukaryotic Protein Interactome

Protein interaction networks are important for the understanding of regulatory mechanisms, for the explanation of experimental data and for the prediction of protein functions. Unfortunately, most interaction data is available only for model organisms. As a possible remedy, the transfer of interactions to organisms of interest is common practice, but it is not clear when interactions can be transferred from one organism to another and, thus, the confidence in the derived interactions is low. Here, we propose to use a rich set of features to train Random Forests in order to score transferred interactions. We evaluated the transfer from a range of eukaryotic organisms to S. cerevisiae using orthologs. Directly transferred interactions to S. cerevisiae are on average only 24% consistent with the current S. cerevisiae interaction network. By using commonly applied filter approaches the transfer precision can be improved, but at the cost of a large decrease in the number of transferred interactions. Our Random Forest approach uses various features derived from both the target and the source network as well as the ortholog annotations to assign confidence values to transferred interactions. Thereby, we could increase the average transfer consistency to 85%, while still transferring almost 70% of all correctly transferable interactions. We tested our approach for the transfer of interactions to other species and showed that our approach outperforms competing methods for the transfer of interactions to species where no experimental knowledge is available. Finally, we applied our predictor to score transferred interactions to 83 targets species and we were able to extend the available interactome of B. taurus, M. musculus and G. gallus with over 40,000 interactions each. Our transferred interaction networks are publicly available via our web interface, which allows to inspect and download transferred interaction sets of different sizes, for various species, and at specified expected precision levels. Availability: http://services.bio.ifi.lmu.de/coin-db/.     

PSIbase: a database of Protein Structural Interactome map (PSIMAP)

Protein Structural Interactome map (PSIMAP) is a global interaction map that describes domain-domain and protein-protein interaction information for known Protein Data Bank structures. It calculates the Euclidean distance to determine interactions between possible pairs of structural domains in proteins. PSIbase is a database and file server for protein structural interaction information calculated by the PSIMAP algorithm. PSIbase also provides an easy-to-use protein domain assignment module, interaction navigation and visual tools. Users can retrieve possible interaction partners of their proteins of interests if a significant homology assignment is made with their query sequences. AVAILABILITY: http://psimap.org and http://psibase.kaist.ac.kr/     

Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a K_D of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The K_D value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo.     

Structural Insights into Yeast Telomerase Recruitment to Telomeres

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A Technique for High-Throughput Protein Crystallization in Ionically Cross-Linked Polysaccharide Gel Beads for X-Ray Diffraction Experiments

A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and κ-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K₂PtBr₆ within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments.     

Discovery of a Non-Peptidic Inhibitor of West Nile Virus NS3 Protease by High-Throughput Docking

Background

The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections.

Methodology

In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by ¹⁵N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay.

Conclusion

The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.     

Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.     

Structural Insights into Alternate Aggregated Prion Protein Forms

The conversion of the cellular form of the prion protein (PrP^C) to an abnormal, alternatively folded isoform (PrP^Sc) is the central event in prion diseases or transmissible spongiform encephalopathies. Recent studies have demonstrated de novo generation of murine prions from recombinant prion protein (recPrP) after inoculation into transgenic and wild-type mice. These so-called synthetic prions lead to novel prion diseases with unique neuropathological and biochemical features. Moreover, the use of recPrP in an amyloid seeding assay can specifically detect and amplify various strains of prions. We employed this assay in our experiments and analyzed in detail the morphology of aggregate structures produced under defined chemical constraints. Our results suggest that changes in the concentration of guanidine hydrochloride can lead to different kinetic traces in a typical thioflavin T(ThT) assay. Morphological and structural analysis of these aggregates by atomic force microscopy indicates a variation in the structure of the PrP molecular assemblies.In particular, ThT positive PrP aggregates produced from rec mouse PrP residues 89 to 230 lead to mostly oligomeric structures at low concentrations of guanidine hydrochloride, while more amyloidal structures were observed at higher concentrations of the denaturant. These findings highlight the presence of numerous and complex pathways in deciphering prion constraints for infectivity and toxicity.     

The Analysis of Participation of Individual Proteins in the Protein Interactome Formation

It becomes increasingly clear that most proteins of living systems exist as components of various protein complexes rather than individual molecules. The use of various proteomic techniques significantly extended our knowledge not only about functioning of individual complexes but also formed a basis for systemic analysis of protein-protein interactions. In this study gel-filtration chromatography accompanied by mass spectrometry was used for the interactome analysis of human liver proteins. In six fractions (with average molecular masses of 45 kDa, 60 kDa, 85 kDa, 150 kDa, 250 kDa, and 440 kDa) 797 proteins were identified. In dependence of their distribution profiles in the fractions, these proteins could be subdivided into four groups: (1) single monomeric proteins that are not involved in formation of stable protein complexes; (2) proteins existing as homodimers or heterodimers with comparable partners; (3) proteins that are partially exist as monomers and partially as components of protein complexes; (4) proteins that do not exist in the monomolecular state, but also exist within protein complexes containing three or more subunits. Application of this approach to known isatin-binding proteins resulted in identification of proteins involved in formation of the homo- and heterodimers and mixed protein complexes.     

Structural Insight into Caenorhabditis elegans Sex-determining Protein FEM-2

In the nematode Caenorhabditis elegans, fem-1, fem-2, and fem-3 play crucial roles in male sexual development. Among these three genes, fem-2 encodes a PP2C (serine/threonine phosphatase type 2C)-like protein, whose activity promotes the development of masculinity. Different from the canonical PP2Cs, FEM-2 consists of an additional N-terminal domain (NTD) apart from its C-terminal catalytic domain. Interestingly, genetic studies have indicated indispensable roles for both of these two domains of FEM-2 in promoting male development, but the underlying mechanism remains unknown. In the present study, we solved the crystal structure of full-length FEM-2, which revealed a novel structural fold formed by its NTD. Structural and functional analyses demonstrated that the NTD did not directly regulate the in vitro dephosphorylation activity of FEM-2, but instead functioned as a scaffold domain in the assembly of the FEM-1/2/3 complex, the executioner in the final step of the sex determination pathway. Biochemical studies further identified the regions in the NTD involved in FEM-1 and FEM-3 interactions. Our results not only identified a novel fold formed by the extra domain of a noncanonical PP2C enzyme, but also provided important insights into the molecular mechanism of how the NTD works in mediating the sex-determining role of FEM-1/2/3 complex.     

Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.