Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity

Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP+ reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP+ binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD+. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD+/H. However, in most cases the analyzed mutants lost the ability for NADP+/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD+/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process.     

The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties

C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 A of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.     

Interaction of nucleoplasmin with core histones

Nucleoplasmin is one of the most abundant proteins in Xenopus laevis oocytes, and it has been involved in the chromatin remodeling that takes place immediately after fertilization. This molecule has been shown to be responsible for the removal of the sperm-specific proteins and deposition of somatic histones onto the male pronuclear chromatin. To better understand the latter process, we have used sedimentation velocity, sedimentation equilibrium, and sucrose gradient fractionation analysis to show that the pentameric form of nucleoplasmin binds to a histone octamer equivalent consisting of equal amounts of the four core histones, H2A, H2B, H3, and H4, without any noticeable preference for any of these proteins. Removal of the histone N-terminal "tail" domains or the major C-terminal polyglutamic tracts of nucleoplasmin did not alter these binding properties. These results indicate that interactions other than those electrostatic in nature (likely hydrophobic) also play a critical role in the formation of the complex between the negatively charged nucleoplasmin and positively charged histones. Although the association of histones with nucleoplasmin may involve some ionic interactions, the interaction process is not electrostatically driven.     

Domain structure characterization of the multifunctional alpha-aminoadipate reductase from Penicillium chrysogenum by limited proteolysis. Activation of alpha-aminoadipate does not require the peptidyl carrier protein box or the reduction domain

The alpha-aminoadipate reductase (alpha-AAR) of Penicillium chrysogenum, an enzyme that activates the alpha-aminoadipic acid by forming an alpha-aminoadipyl adenylate and reduces the activated intermediate to alpha-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for alpha-aminoadipic acid, ATP, and NADPH were determined. Sequencing of the N-terminal end confirmed the 10 first amino acids deduced from the nucleotide sequence. Its domain structure has been investigated using limited proteolysis and active site labeling. Trypsin and elastase were used to cleave the multienzyme, and the location of fragments within the primary structure was established by N-terminal sequence analysis. Initial proteolysis generated two fragments: an N-terminal fragment housing the adenylation and the peptidyl carrier protein (PCP) domains (116 kDa) and a second fragment containing most of the reductive domain (28 kDa). Under harsher conditions the adenylation domain (about 64 kDa) and the PCP domain (30 kDa) become separated. Time-dependent acylation of alpha-AAR and of fragments containing the adenylation domain with tritiated alpha-aminoadipate occurred in vitro in the absence of NADPH. Addition of NADPH to the labeled alpha-AAR released most of the radioactive substrate. A fragment containing the adenylation domain was labeled even in absence of the PCP box. The labeling of this fragment (lacking PCP) was always weaker than that observed in the di-domain (adenylating and PCP) fragment suggesting that the PCP domain plays a role in the stability of the acyl intermediate. Low intensity direct acylation of the PCP box has also been observed. A domain structure of this multienzyme is proposed.     

Dimers of class A G protein-coupled receptors function via agonist-mediated trans-activation of associated G proteins

The histamine H1 receptor and the alpha1b-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca2+] via activation of Gq/G11. Assessed by co-immunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homo-dimers. The addition of the G protein G11alpha to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5'-3-O-([35S]thio)triphosphate ([35S]GTPgammaS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG11alpha into the fusions. Mutation of a highly conserved leucine in intracellular loop 2 of each receptor also eliminated agonist function but not binding. Co-expression of the two non-functional but complementary fusion constructs reconstituted agonist-mediated binding of [35S]GTPgammaS in membranes of HEK293 cells and elevation of [Ca2+]i in mouse embryo fibroblasts lacking both Gq and G11. Co-expression of the histamine H1 receptor- and the alpha1b-adrenoreceptor-G11alpha fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G11alpha with increasing amounts of L151Dalpha1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [35S]GTPgammaS binding. Co-expression of the alpha1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the alpha1b-adrenoreceptor and G11alpha did not result in agonist activation of the G protein but did indicate a role for transmembrane helix 1 in dimerization. These data demonstrate that dimers of these class A receptors function via trans-activation of associated G proteins.     

MAL regulates clathrin-mediated endocytosis at the apical surface of Madin-Darby canine kidney cells

MAL is an integral protein component of the machinery for apical transport in epithelial Madin-Darby canine kidney (MDCK) cells. To maintain its distribution, MAL cycles continuously between the plasma membrane and the Golgi complex. The clathrin-mediated route for apical internalization is known to differ from that at the basolateral surface. Herein, we report that MAL depends on the clathrin pathway for apical internalization. Apically internalized polymeric Ig receptor (pIgR), which uses clathrin for endocytosis, colocalized with internalized MAL in the same apical vesicles. Time-lapse confocal microscopic analysis revealed cotransport of pIgR and MAL in the same endocytic structures. Immunoelectron microscopic analysis evidenced colabeling of MAL with apically labeled pIgR in pits and clathrin-coated vesicles. Apical internalization of pIgR was abrogated in cells with reduced levels of MAL, whereas this did not occur either with its basolateral entry or the apical internalization of glycosylphosphatidylinositol-anchored proteins, which does not involve clathrin. Therefore, MAL is critical for efficient clathrin-mediated endocytosis at the apical surface in MDCK cells.     

Drought enhances maize chilling tolerance. II. Photosynthetic traits and protective mechanisms against oxidative stress

In the present research we studied the photosynthetic traits and protective mechanisms against oxidative stress in two maize ( Zea mays L.) genotypes differing in chilling sensitivity (Z7, tolerant and Penjalinan, sensitive) subjected to 5°C for 5 days, with or without pretreatment by drought. The drought pretreatment decreased the symptoms of chilling injury in Penjalinan plants estimated as necrotic leaf area and maximum quantum yield of photosystem II. Furthermore, drought pretreatment diminished the level of lipid peroxidation caused by chilling in Penjalinan plants. After one day of recovery from chilling the Z7 and drought-pretreated Penjalinan plants showed higher net photosynthesis rates than the non-drought-pretreated Penjalinan plants, thereby decreasing the probability of generating reactive oxygen species. The greater net photosynthesis was correlated with the greater NADP-malate dehydrogenase activity. No differences in either the de-epoxidation state of the xanthophyll cycle or the antioxidant enzyme activities were found among the chilled groups of plants. However, a drastic decrease in ascorbate content was observed in chilled Penjalinan plants without drought pretreatment. As we found an increase of H₂O₂ content after drought pretreatment, we suggest its involvement as a signal in the drought-enhanced chilling tolerance of maize.     

Bacterial host strains that support replication of somatic coliphages

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log₁₀ fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible. This revised version was published online in June 2006 with corrections to the Cover Date.