Amino acid substitutions at protein–protein interfaces that modulate the oligomeric state

Despite similarities in their sequence and structure, there are a number of homologous proteins that adopt various oligomeric states. Comparisons of these homologous protein pairs, in terms of residue substitutions at the protein–protein interfaces, have provided fundamental characteristics that describe how proteins interact with each other. We have prepared a dataset composed of pairs of related proteins with different homo‐oligomeric states. Using the protein complexes, the interface residues were identified, and using structural alignments, the shadow‐interface residues have been defined as the surface residues that align with the interface residues. Subsequently, we investigated residue substitutions between the interfaces and the shadow interfaces. Based on the degree of the contributions to the interactions, the aligned sites of the interfaces and shadow interfaces were divided into primary and secondary sites; the primary sites are the focus of this work. The primary sites were further classified into two groups (i.e. exposed and buried) based on the degree to which the residue is buried within the shadow interfaces. Using these classifications, two simple mechanisms that mediate the oligomeric states were identified. In the primary‐exposed sites, the residues on the shadow interfaces are replaced by more hydrophobic or aromatic residues, which are physicochemically favored at protein–protein interfaces. In the primary‐buried sites, the residues on the shadow interfaces are replaced by larger residues that protrude into other proteins. These simple rules are satisfied in 23 out of 25 Structural Classification of Proteins (SCOP) families with a different‐oligomeric‐state pair, and thus represent a basic strategy for modulating protein associations and dissociations. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Does any yeast mitochondrial carrier have a native uncoupling protein function?

In this study, we explore the hypothesis that some member of the mitochondrial carrier family has specific uncoupling activity that is responsible for the basal proton conductance of mitochondria. Twenty-seven of the 35 yeast mitochondrial carrier genes were independently disrupted in Saccharomyces cerevisiae. Six knockout strains did not grow on nonfermentable carbon sources such as lactate. Mitochondria were isolated from the remaining 21 strains, and their proton conductances were measured. None of the 21 carriers contributed significantly to the basal proton leak of yeast mitochondria. A possible exception was the succinate/fumarate carrier encoded by the Xc2 gene, but deletion of this gene also affected yeast growth and respiratory chain activity, suggesting a more general alteration in mitochondrial function. If a specific protein is responsible for the basal proton conductance of yeast mitochondria, its identity remains unknown.     

Evolution and diversity of axon guidance Robo receptor family genes

The diversity of axon guidance (AG) receptors reflects gains in complexity of the animal nervous system during evolution. Members of the Roundabout (Robo) family of receptors interact with Slit proteins and play important roles in many developmental processes, including AG and neural crest cell migration. There are four members of the Robo gene family. However, the evolutionary history of Robo family genes remain obscure. We analyzed the distribution of Robo family members in metazoan species ranging in complexity from hydras to humans. We undertook a phylogenetic analysis in metazoans, synteny analysis, and ancestral chromosome mapping in vertebrates, and detected selection pressure and functional divergence among four mammalian Robo paralogs. Based on our analysis, we proposed that the ancestral Robo gene could have undergone a tandem duplication in the vertebrate ancestor; then one round of whole genome duplication events occurred before the divergence of ancestral lamprey and gnathostome, generating four paralogs in early vertebrates. Robo4 paralog underwent segmental loss in the following evolutionary process. Our results showed that Robo3 paralog is under more powerful purifying selection pressure compared with other three paralogs, which could correlate with its unique expression pattern and function. Furthermore, we found four sites under positive selection pressure on the Ig1‐2 domains of Robo4 that might interfere with its binding to Slits ligand. Diverge analysis at the amino acid level showed that Robo4 paralog have relatively greater functional diversifications than other Robo paralogs. This coincides with the fact that Robo4 predominantly functions in vascular endothelial cells but not the nervous system.     

The structural analysis and the role of calcium binding site for thermal stability in mannanase

Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K_a of 3.02 × 10⁴ M⁻¹ and 1.52 × 10⁴ M⁻¹, respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.     

G1 cell cycle arrest due to the inhibition of erbB family receptor tyrosine kinases does not require the retinoblastoma protein

The erbB receptor family (EGFr, erbB-2, erbB-3, and erbB-4) consists of transmembrane glycoproteins that transduce extracellular signals to the nucleus when activated. erbB family members are widely expressed in epithelial, mesenchymal, and neuronal cells and contribute to the proliferation, differentiation, migration, and survival of these cell types. The present study evaluates the effects of erbB family signaling on cell cycle progression and the role that pRB plays in regulating this process. ErbB family RTK activity was inhibited by PD 158780 in the breast epithelial cell line MCF10A. PD 158780 (0.5 microM) inhibited EGF-stimulated and heregulin-stimulated autophosphorylation and caused a G1 cell cycle arrest within 24 h, which correlated with hypophosporylation of pRB. MCF10A cells lacking functional pRB retained the ability to arrest in G1 when treated with PD 158780. Both cell lines showed induction of p27(KIP1) protein when treated with PD 158780 and increased association of p27(KIP1) with cyclin E-CDK2. Furthermore, CDK2 kinase activity was dramatically inhibited with drug treatment. Changes in other pRB family members were noted with drug treatment, namely a decrease in p107 and an increase in p130. These findings show that the G1 arrest induced through inhibition of erbB family RTK activity does not require functional pRB.     

Synthesis of Guanosine-3′-diphosphate- 5′-diphosphate by Nucleotide Pyrophosphotransferase

An α-glucosidase was purified from sweet corn seeds by fractionation with ammonium sulfate, chromatographies on CM-Sepharose and Sepharose 4B, and gel filtrations on Sephadex G-100. The enzyme was homogeneous in disc electrophoretic analysis. The molecular weight was estimated to be about 9.6 × 10⁴ by SDS-disc electrophoresis.

The enzyme showed high activities toward maltose, nigerose, phenyl-α-maltoside, and maltooligosaccharides. The ratios of maximum velocity for maltose, nigerose, kojibiose, isomaltose, phenyl-α-glucoside, phenyl-α-maltoside, panose, turanose, and soluble starch were estimated to be 100 : 78 : 17 : 11 : 28 : 100 : 31 : 3.4 : 126, and the Km values for these substrates, 1.5 mM, 1.4 mM, 0.48 mM, 14 mM, 4.2 mM, 1.1 mM, 5.0 mM, 0.28 mM and 52mg/ml, respectively. The maximum velocity for soluble starch was high, but this α-glucan was not a favorable substrate because the Km value was also very high. The V_max for maltooligosaccharides were somewhat dependent on the degree of polymerization (n). The Km values for substrates having four or more glucose units increased with the increase in n.     

Chromosome-level genome of Tibetan naked carp (Gymnocypris przewalskii) provides insights into Tibetan highland adaptation

Gymnocypris przewalskii, a cyprinid fish endemic to the Qinghai-Tibetan Plateau, has evolved unique morphological, physiological and genetic characteristics to adapt to the highland environment. Herein, we assembled a high-quality G. przewalskii tetraploid genome with a size of 2.03 Gb and scaffold N50 of 44.93 Mb, which was anchored onto 46 chromosomes. The comparative analysis suggested that gene families related to highland adaptation were significantly expanded in G. przewalskii. According to the G. przewalskii genome, we evaluated the phylogenetic relationship of 13 schizothoracine fishes, and inferred that the demographic history of G. przewalskii was strongly associated with geographic and eco-environmental alterations. We noticed that G. przewalskii experienced whole-genome duplication, and genes preserved post duplication were functionally associated with adaptation to high salinity and alkalinity. In conclusion, a chromosome-scale G. przewalskii genome provides an important genomic resource for teleost fish, and will particularly promote our understanding of the molecular evolution and speciation of fish in the highland environment.