Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%–75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.     

Stem and crown allometry in four congeneric species of dioecious tropical trees

Key message

Congeneric species showed similar stem and crown allometry, but differed in crown dimensions indicating that crown size is adaptive and variable despite mechanical restrictions.

Abstract

Morphological adaptations favor differential use of the space in tropical trees, but the variability in stem and crown allometry can be constrained by phylogenetic and mechanical factors. In addition, dioecious species show marked differences in their energy requirements related to reproduction, but little information is available about the role of shape and allometry on differential acquisition of energy between the sexes. We studied the stem and crown dimensions of congeneric dioecious trees to determine if there are: (i) differences in the allometry between the sexes, (ii) different average sizes among sympatric species, and (iii) differences in stem and crown allometry between sympatric and allopatric species. Two pairs of sympatric Virola (Myristicaceae) in Brazil and Costa Rica were studied. SMA regression models were used to investigate allometric relationships between diameter at breast height (DBH) and tree height, and between DBH and crown volume (CV). No sexual dimorphism in stem and crown morphology was observed in this study, indicating that differences in resource allocation for reproduction between the sexes do not impact the stem and crown structure in these species. Overall, low variability among the species was observed. Only one species differed in stem allometry and none differed in crown allometry. CV differed between sympatric species. Stem and crown allometry are related to structural stability and our results support similar mechanical restriction for these species. The ecological significance of differences in CV among canopy species remains to be explored.

     

Using experimental evolution to probe molecular mechanisms of protein function

Directed evolution is a powerful tool for engineering protein function. The process of directed evolution involves iterative rounds of sequence diversification followed by assaying activity of variants and selection. The range of sequence variants and linked activities generated in the course of an evolution are a rich information source for investigating relationships between sequence and function. Key residue positions determining protein function, combinatorial contributors to activity and even potential functional mechanisms have been revealed in directed evolutions. The recent application of high throughput sequencing substantially increases the information that can be retrieved from directed evolution experiments. Combined with computational analysis this additional sequence information has allowed high‐resolution analysis of individual residue contributions to activity. These developments promise to significantly enhance the depth of insight that experimental evolution provides into mechanisms of protein function.     

Polymorphism and complexity of HLA-DR: evidence for intra-HLA-DR region crossing-over events

HLA-DR molecules were isolated from HLA-DR3, –5, and –w6 positive homozygous B-cell lines by immunoprecipitation with monoclonal antibodies and analyzed by gel electrophoretic techniques. DNA isolated from the same cell lines was digested with the restriction enzyme Taq I and hybridized with a DR beta full-length cDNA probe. We demonstrated that certain DR I alleles are found in combination with different DR III alleles as defined by Southern blotting, protein chemistry, a functional assay using purified protein derivative-specific T-cell lines, and, in one case, also alloreactive T-cell reagents. Our results indicate that within the family of HLA-DRw52-associated haplotypes DR beta chain genes may have been transferred from one haplotype to another. The implications of these findings are discussed.     

Ultrastructure and adrenergic innervation of preglomerular arterioles in the euryhaline teleost,Salmo gairdneri

Summary The opisthonephric kidney of the rainbow trout was investigated by light- and electron microscopy and a fluorescent-histochemical technique for biogenic amines was used. Preglomerular sphincters at the origin of afferent arterioles are present in this euryhaline teleost. The branching point of the afferent arteriole is characterized by (i) the formation of a right angle with the parent vessel, (ii) circularly arranged smooth muscle cells of the tunica media, (iii) additional circularly arranged smooth muscle cells intercalated between endothelium and tunica media, and (iv) a collar-like arrangement of several large endothelial cells with elaborate marginal folds and abundant myoendothelial junctions. A dense adrenergic innervation displaying specific fluorescence was found along the terminal arterioles and afferent arterioles, and conspicuously at the preglomerular sphincters. These results are suggestive of a neural participation in kidney function. They are discussed on the basis of recent evidence from pharmacological and physiological experiments for neural involvement in glomerular intermittency.