Asparaginyl-tRNA synthetase from the Escherichia coli temperature-sensitive strain HO202 A proline replacement in motif 2 is responsible for a large increase in Km for asparagine and ATP

AbstractThe Escherichia coli K12 mutant gene, asnS40, coding for asparaginyl-tRNA synthetase (AsnRS) in the temperature-sensitive strain HO202, was isolated from genomic DNA using the Polymerase Chain Reaction. DNA sequencing revealed that the mutant enzyme differs from the wild-type AsnRS by two amino acids, but only the P231L replacement leads to a change in aminoacylation activity. In the ATP-PPi exchange reaction at 37°C the purified P231L enzyme has a more than 50-fold increased Km value for asparagine compared to the wild-type enzyme, while the Km value for ATP is increased 8-fold. In the aminoacylation reaction the mutant enzyme shows also significantly increased Km values for asparagine and ATP. Interestingly Pro-231 is part of the conserved motif 2 in class II aminoacyl-tRNA synthetases (Eriani, G., Delarue, M., Poch, O., Gangloff, J. and Moras, D. (1990) Nature 347, 203–206), indicating that this motif might be involved in all class II enzymes in amino acid activation

Structural studies of hydrated samples of amorphous calcium phosphate and phosphoprotein nanoclusters

There are abundant examples of nanoclusters and inorganic microcrystals in biology. Their study under physiologically relevant conditions remains challenging due to their heterogeneity, instability, and the requirements of sample preparation. Advantages of using neutron diffraction and contrast matching to characterize biomaterials are highlighted in this article. We have applied these and complementary techniques to search for nanocrystals within clusters of calcium phosphate sequestered by bovine phosphopeptides, derived from osteopontin or casein. The neutron diffraction patterns show broad features that could be consistent with hexagonal hydroxyapatite crystallites smaller than 18.9 Å. Such nanocrystallites are, however, undetected by the complementary X-ray and FTIR data, collected on the same samples. The absence of a distinct diffraction pattern from the nanoclusters supports the generally accepted amorphous calcium phosphate structure of the mineral core

Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

Autoantibodies to histidyl–tRNA synthetase (HisRS) or to alanyl–, asparaginyl–, glycyl–, isoleucyl–, or threonyl–tRNA synthetase occur in ∼25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl–tRNA synthetases to induce leukocyte migration. HisRS induced CD4+ and CD8+ lymphocytes, interleukin (IL)-2–activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH2-terminal domain, 1–48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl–tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl–tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl–tRNA and lysyl–tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl–tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself

Transport of hemolysin by Escherichia coli

No abstract availabl

Effect of phosphorylation on a human-like osteopontin peptide

The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals