A proximal arginine R206 participates in switching of the Bradyrhizobium japonicum FixL oxygen sensor

In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd approximately 350 microM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the Fe(III) form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.     

The VIP-receptor system in neuroblastoma cells

Neuroblastoma (NB), the most common extracranial tumor during childhood arises from the embryonic sympathetic nervous system. Remarkably, NB can spontaneously regress, even after metastasis, leading to complete remission. Subpopulations of neuroblastic (N-type) and nonneuronal cells coexist in NB. Expression of the high-affinity nerve growth factor (NGF) TrkA receptor in NB is correlated with good prognosis, while MYCN amplification is associated with advanced stages of disease. N-type cells undergo differentiation when treated with different compounds, such as retinoids, phorbol esters, growth and neurotrophic NGF and neuropeptides, especially vasoactive intestinal peptide (VIP). These substances stabilize proliferation, leading to a more mature neuronal phenotype, neurite outgrowth and induction of expression of sympathetic neuronal markers. Therefore, receptors for these substances and their associated signalling pathways, appear like promising targets for the development of novel NB therapeutics. The aim of the present review is to summarize the quite considerable array of data, concerning production of VIP and related peptides, expression of their receptors in NB and the key regulation exerted by the VIP-receptor system in the control of NB cell behaviour.     

The use of random controls in genetic association studies

BACKGROUND: The optimal control sample would be ethnically-matched and at minimal risk of developing the disease. Alternatively, one could collect random individuals from the population or select individuals to reduce the number of at-risk individuals in the sample. The effect of randomly selected individuals in a control sample on the statistical power and the odds ratio estimate was investigated. METHODS: Case and control genotype distributions were simulated using standard genetic models with an additional term representing the proportion of unidentified cases in the control sample. Power and odds ratio were calculated from the genotype distributions generated under different sampling scenarios using established methods. RESULTS: Random sampling of controls resulted in a loss in power and a reduction in the odds ratio estimate to a degree that is determined by the proportion of random sampling and the prevalence of the disease. Random sampling resulted in a 19% loss in power for a disease having prevalence of 0.20, compared to a control sample that contained no at-risk individuals. Having random controls results in a decrease in the odds ratio estimate. CONCLUSIONS: Investigators planning case-control genetic association studies should be aware of the statistical costs of different ascertainment approaches.     

Changes at the KinA PAS-A dimerization interface influence histidine kinase function

The Bacillus subtilis KinA protein is a histidine protein kinase that controls the commitment of this organism to sporulate in response to nutrient deprivation and several other conditions. Prior studies indicated that the N-terminal Per-ARNT-Sim domain (PAS-A) plays a critical role in the catalytic activity of this enzyme, as demonstrated by the significant decrease of the autophosphorylation rate of a KinA protein lacking this domain. On the basis of the environmental sensing role played by PAS domains in a wide range of proteins, including other bacterial sensor kinases, it has been suggested that the PAS-A domain plays an important regulatory role in KinA function. We have investigated this potential by using a combination of biophysical and biochemical methods to examine PAS-A structure and function, both in isolation and within the intact protein. Here, we present the X-ray crystal structure of the KinA PAS-A domain, showing that it crystallizes as a homodimer using beta-sheet/beta-sheet packing interactions as observed for several other PAS domain complexes. Notably, we observed two dimers with tertiary and quaternary structure differences in the crystalline lattice, indicating significant structural flexibility in these domains. To confirm that KinA PAS-A also forms dimers in solution, we used a combination of NMR spectroscopy, gel filtration chromatography, and analytical ultracentrifugation, the results of which are all consistent with the crystallographic results. We experimentally tested the importance of several residues at the dimer interface using site-directed mutagenesis, finding changes in the PAS-A domain that significantly alter KinA enzymatic activity in vitro and in vivo. These results support the importance of PAS domains within KinA and other histidine kinases and suggest possible routes for natural or artificial regulation of kinase activity.