Crystal structures of the copper and nickel complexes of RNase A: Metal-induced interprotein interactions and identification of a novel copper binding motif

We report the crystal structures of the copper and nickel complexes of RNase A. The overall topology of these two complexes is similar to that of other RNase A structures. However, there are significant differences in the mode of binding of copper and nickel. There are two copper ions per molecule of the protein, but there is only one nickel ion per molecule of the protein. Significant changes occur in the interprotein interactions as a result of differences in the coordinating groups at the common binding site around His-105. Consequently, the copper- and nickel-ion-bound dimers of RNase A act as nucleation sites for generating different crystal lattices for the two complexes. A second copper ion is present at an active site residue His-119 for which all the ligands are from one molecule of the protein. At this second site, His-119 adopts an inactive conformation (B) induced by the copper. We have identified a novel copper binding motif involving the α-amino group and the N-terminal residues.

Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1 gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants

A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants. To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus. Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni2+-NTA-agarose (NTA is nitrilotriacetic acid). Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations. LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments. Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII. This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast. Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-Å resolution. LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins. This mutant was monomeric in the photosynthetic membrane probably due to the deletion of the phosphatidylglycerol binding site and was degraded by the plastidic proteolytic system. Exchange of Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably due to the lack of a chlorophyll a binding site.

Polypeptides of the Maize Amyloplast Stroma1 : Stromal Localization of Starch-Biosynthetic Enzymes and Identification of an 81-Kilodalton Amyloplast Stromal Heat-Shock Cognate

In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

Isolation and identification of a diuretic hormone from the mealworm Tenebrio molitor.

A diuretic hormone of unusual structure was isolated from extracts of whole heads of the mealworm Tenebrio molitor. The hormone is a 37-aa peptide of 4371 Da, with the sequence SPTISITAPIDVLRKTWEQERARKQMVKNREFLNSLN. This peptide increases cAMP production in Malpighian tubules of T. molitor. The amino acid sequence reveals that this peptide is a member of the family of sauvagine/corticotropin-releasing factor/urotensin I-related insect diuretic hormones. The C-terminal sequence of this peptide is quite different from other members of this family, which have a hydrophobic C terminus (isoleucinamide or valinamide). When aligned comparably, T. molitor diuretic hormone has a more hydrophilic C terminus, leucylasparagine (free acid). In contrast to all other known diuretic hormones of this family, this peptide has exceptionally low stimulatory activity on cAMP production in Malpighian tubules of Manduca sexta. However, at nanomolar concentrations it stimulates cAMP production in Malpighian tubules of T. molitor. Diuretic hormones of this family have been isolated previously from Lepidoptera, Orthoptera, Dictyoptera, and Diptera. This appears to be the first diuretic hormone isolated from a coleopteran insect.

Purification and identification of pituitary cytotropic factor.

It has been shown that the pituitary contains a cytotropic factor (CTF) that stimulates the secretion of catecholamines by dopaminergic neurons of the hypothalamus. In the present study, CTF was purified from rat pituitaries and found by means of mass spectrometric analysis to be adenosine. This finding was corroborated by the observations that CTF behaves identically to adenosine when subjected to liquid chromatography, is inactivated and converted to inosine by adenosine deaminase, and is qualitatively and quantitatively indistinguishable from adenosine in its biological activity. It is concluded that pituitary adenosine is a trophic factor for hypothalamic dopaminergic neurons.

Physical mapping, cloning, and identification of genes within a 500-kb region containing BRCA1.

BRCA1 is a breast/ovarian cancer susceptibility gene on human chromosome 17q21. We describe a complete and detailed physical map of a 500-kb region of genomic DNA containing the BRCA1 gene and the partial cloning in phage P1 artificial chromosomes. Approximately 70 exons were isolated from this region, 11 of which were components of the BRCA1 gene. Analysis of the other exons revealed a rho-related G protein and the interferon-induced leucine-zipper protein IFP-35.

Motorcycle accident cause factors and identification of countermeasures. Volume II: appendix/supplemental data. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Motorcycle accident cause factors and identification of countermeasures. Volume I: technical report. Final report.

National Highway Traffic Safety Administration, Washington, D.C.