Structural and functional analyses of PpENA1 provide insights into cation binding by type IID P-type ATPases in lower plants and fungi

PpENA1 is a membrane-spanning transporter from the moss Physcomitrella patens, and is the first type IID P-type ATPase to be reported in the plant kingdom. In Physcomitrella, PpENA1 is essential for normal growth under moderate salt stress, while in yeast, type IID ATPases provide a vital efflux mechanism for cells under high salt conditions by selectively transporting Na+ or K+ across the plasma membrane. To investigate the structural basis for cation-binding within the type IID ATPase subfamily, we used homology modeling to identify a highly conserved cation-binding pocket between membrane helix (MH) 4 and MH 6 of the membrane-spanning pore of PpENA1. Mutation of specific charged and polar residues on MHs 4-6 resulted in a decrease or loss of protein activity as measured by complementation assays in yeast. The E298S mutation on MH 4 of PpENA1 had the most significant effect on activity despite the presence of a serine at this position in fungal type IID ATPases. Activity was partially restored in an inactivated PpENA1 mutant by the insertion of two additional serine residues on MH 4 and one on MH 6 based on the presence of these residues in fungal type IID ATPases. Our results suggest that the residues responsible for cation-binding in PpENA1 are distinct from those in fungal type IID ATPases, and that a fungal-type cation binding site can be successfully engineered into the moss protein.     

Structural and functional relationships of FAN1

FANCD2/FANCI-associated nuclease (FAN1) is a 5′ flap structure-specific endonuclease and 5′ to 3′ exonuclease. This nuclease can resolve interstrand cross-links (ICLs) independently of the Fanconi anemia (FA) pathway and controls the progression of stalled replication forks in an FA-dependent manner, thereby maintaining chromosomal stability. Several FAN1 mutations are observed in various cancers and degenerative diseases. Recently, several crystal structures of the FAN1-DNA complexes have been reported, and to date, these represent the only structures for a DNA bound ICL-repair nuclease. Puzzlingly, human FAN1 forms two different quaternary structures with different DNA binding modes, and based on these structures, two ICL-repair mechanisms have been proposed. In one mechanism, monomeric FAN1 recognizes the 5′ flap terminal phosphate via a basic pocket and successively cleaves at every third nucleotide of the DNA substrates. In the other mechanism, dimeric FAN1 scans, latches, and unwinds the postnick duplex of the substrate DNA to direct the scissile phosphodiester group to the active site. In this review, we discuss the structures, function, and proposed mechanisms of FAN1 nuclease, and provide the insights into its role in ICL repair and in processing of stalled replication forks.     

Structural and functional relationships between aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases can be divided in two groups of equal size on the basis of differences in the structure of their active sites. The core of class I synthetases is the classical nucleotide-binding domain with its characteristic Rossmann fold. In contrast, the active site of class II synthetases is built around an antiparallel beta-sheet, to which the substrates bind. This classification, which is based on structural data (amino acid sequences and tertiary structures), can be rationalized in functional terms.     

Structural and functional relationships between h- and l-caldesmons.

Two different Mr forms of caldesmon as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr values in the range of 120,000-150,000, h-caldesmon and 70,000-80,000, l-caldesmon) have been already identified. h-Caldesmon is predominantly expressed in smooth muscle cells, whereas l-caldesmon widely distributes in non-muscle cells. Most recently, the molecular cloning of h-caldesmon has been reported (Hayashi, K., Kanda, K., Kimizuka, F., Kato, I., and Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164, 503-511; Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W-G. (1989) J. Biol. Chem. 264, 13873-13879). The calculated Mr of this protein from its primary structure is 88,743. Here, the nucleotide and deduced amino acid sequences of l-caldesmon have been determined by cloning and sequencing the cDNA from chick brain and compared with those of h-caldesmon. The l-caldesmon cDNA encodes a sequence of 517 amino acids with the calculated Mr of 58,844. Two isoforms of caldesmon conserve the completely identical sequences in the NH2- and COOH-terminal domains except for the insertion of Ala-508 in l-caldesmon. Interestingly, the central repeating sequence of h-caldesmon (residues 201-447) is deleted in the l-caldesmon molecule. The short NH2-terminals of two caldesmons individually show the unique sequences. The results of Northern and Southern blot analyses suggest that two mRNAs (4.8 and 4.1 kilo-bases) coding for caldesmon isoforms may be generated from a single gene by alternative splicing. Using a series of truncated caldesmons expressed in Escherichia coli, the common calmodulin-, tropomyosin-, and actin-binding sites and the minimum regulatory domains, which are involved in the Ca2(+)-dependent regulation of actin-myosin interaction, have been identified within the limited consensus sequences (residues 381-433 for l-caldesmon and residues 636-688 for h-caldesmon).     

Mutagenesis and functional analysis of the Escherichia coli tRNALeu1 promoter

The leuV promoter which produces tRNA^Leu₁ in Escherichia coli has been extensively mutagenized in order to determine the effects of altered sequences on promoter efficiency (strength) and on growth-rate-dependent regulation (GDR). Each mutant promoter was ligated with a β-galactosidase reporter gene into the chromosome of a host cell by phage lambda lysogenization. Reporter gene activities were measured for cells growing in selected media at various growth rates. Sequences which flank the ?10 consensus region, when altered, caused remarkable up-promoter effects, increasing efficiency in some cases almost 10-fold. One up mutation which had five successive T residues in the‘discriminator’region completely abolished GDR, whereas several mutations with single base changes in the discriminator had little or no effect on GDR. Another mutation which changed one base in the ?35 region to bring it to consensus increased promoter strength 18-fold and sharply reduced GDR. Chimaeric promoters in which segments of leuV were replaced by segments of the his operon showed that only when the discriminator of leuV is replaced by the his discriminator was GDR-disturbed. AM upstream sequences which were replaced by his sequences had little effect on GDR. Overall, there appeared to be little correlation between promoter efficiency and GDR.     

Atx1-like chaperones and their cognate P-type ATPases: copper-binding and transfer

Copper is an essential yet toxic metal ion. To satisfy cellular requirements, while, at the same time, minimizing toxicity, complex systems of copper trafficking have evolved in all cell types. The best conserved and most widely distributed of these involve Atx1-like chaperones and P_1B-type ATPase transporters. Here, we discuss current understanding of how these chaperones bind Cu(I) and transfer it to the Atx1-like N-terminal domains of their cognate transporter.     

P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Arabidopsis thaliana has eight genes encoding members of the type 1_B heavy metal–transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.     

Distinct functional roles for the Menkes and Wilson copper translocating P-type ATPases in human placental cells.

BACKGROUND/AIMS: The copper transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND) are essential for normal copper transport in the human body. The placenta is the key organ in copper supply to the fetus during pregnancy and it is one of the few organs in the body to express both of the ATPases. The placenta therefore provides a unique opportunity to elucidate the specific roles of these transporters within the one cell type. METHODS/RESULTS: Using polarized placental Jeg-3 cells, siRNA technology and radio-labelled 64Cu transport assays, MNK and WND were shown to have distinct roles in the vectorial transport of copper. MNK transported copper from the cell via the basolateral membrane and in contrast, WND transported copper from the apical membrane. Inactivation of MNK resulted in decreased activity of two important cuproenzymes, lysyl oxidase and Cu/Zn-superoxide dismutase. CONCLUSIONS: Overall, these results provide definitive evidence for distinct roles of MNK and WND in the human placenta, and are consistent with a role for MNK in the transport of copper into the fetal circulation, and through delivery of copper to placental cuproenzymes, whilst WND contributes to the maintenance of placental copper homeostasis by transporting copper to the maternal circulation.     

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent Mr, migrates on SDS-PAGE with an apparent Mr of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent Mr) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent Mr). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.     

Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.     

Solubilization of Serotonin1a and Serotonin1b Binding Sites from Bovine Brain

Serotonin1 (5-hydroxytryptamine1, 5-HT1) binding sites have been solubilized from bovine brain cortex using a mixture of 0.1% Nonidet P-40 and 0.3% digitonin in a low-salt buffer containing 0.1% ascorbic acid. The affinity of [3H]5-HT for the soluble cortical binding sites (2.1 nM) is identical to its affinity at membrane-bound binding sites (2.1 nM). [3H]8-Hydroxy-2-(di-n-propylamino)tetralin ([3H]DPAT), a selective 5-HT1a radioligand, also binds to soluble cortical binding sites with high affinity (1.8 nM) comparable with its affinity in the crude membranes (1.7 nM). A significant correlation exists in the rank order potency of serotonergic agents for [3H]5-HT binding and for [3H]DPAT binding to crude and soluble membranes. The density of [3H]DPAT binding sites relative to the [3H]5-HT sites in the solubilized cortical membranes (35%) corresponds well with the proportion of 5-HT1a sites in the crude membranes determined by spiperone displacement (33%), suggesting that both the 5-HT1a and 5-HT1b binding sites have been cosolubilized. [3H]5-HT binding in the soluble preparations was inhibited by GTP, suggesting that a receptor complex may have been solubilized. [3H]Spiperone-specific binding was not detectable in this preparation, suggesting that 5-HT2 sites were not cosolubilized.     

Elevated CO2 decreases seed germination in Arabidopsis thaliana

The impact of elevated [CO₂] on seed germination was studied in different genotypes of Arabidopsis thaliana from natural populations. Two generations of seeds were studied: the maternal generation was produced in the greenhouse (present-day conditions), the offspring generation was produced in two chambers where the CO₂ concentration was either the present atmospheric concentration (about 350 ppm) or elevated (700 ppm). The seeds were tested for proportion of germinated seeds and mean germination time in both chambers to study the impact of elevated [CO₂] during seed production and germination. Elevated [CO₂] during maturation of seeds on the mother-plants decreased the proportion of germinated seeds, while elevated [CO₂] during germination had no effect on the proportion of germinated seeds. However, when seeds were both produced and germinated under elevated [CO₂] (situation expected by the end of next century), germination was slow and low. Moreover, the effect of the [CO₂] treatment differs among genotypes of Arabidopsis: there is a strong treatment × genotype interaction. This means that there is ample genetic variance for a selective response modiying the effects of high levels of [CO₂] in natural populations of Arabidopsis thaliana. The outcome at the community level will depend on what seeds are available, when they germinate and the resulting competition following germination.     

Structural and functional relationships between the lectin and arm domains of calreticulin

Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a K(d) for peptides of 0.5-1 μM. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function.     

Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases.

The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.     

Pharmacological Differentiation and Characterization of 5-HT1A, 5-HT1B, and 5-HT1C Binding Sites in Rat Frontal Cortex

Drug interactions with 5-HT1 (5-hydroxytryptamine type 1) binding site subtypes were analyzed in rat frontal cortex. 8-Hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) displays high affinity (Ki 3.3 +/- 1 nM) for 29 +/- 3% of total [3H]5-HT binding in rat frontal cortex and low affinity (Ki 9,300 +/- 1,000) for 71 +/- 4% of the remaining 5-HT1 sites. Therefore, non-5-HT1A binding in rat frontal cortex was defined as specific [3H]5-HT binding observed in the presence of 100 nM 8-OH-DPAT. 5-Methoxy 3-(1,2,3,6-tetrahydro-4-pyridinyl) 1 H indole (RU 24969), 1-(m-trifluoromethylphenyl)piperazine (TFMPP), mianserin, and methysergide produce shallow competition curves of [3H]5-HT binding from non-5-HT1A sites. Addition of 10(-3) M GTP does not increase the apparent Hill slopes of these competition curves. Computer-assisted iterative curve fitting suggests that these drugs can discriminate two distinct subpopulations of non-5-HT1A binding sites, each representing approximately 35% of the total [3H]5-HT binding in the rat frontal cortex. All three 5-HT1 binding site subtypes display nanomolar affinity for 5-HT and 5-methoxytryptamine. A homogeneous population of 5-HT1A sites can be directly labeled using [3H]8-OH-DPAT. These sites display nanomolar affinity for 8-OH-DPAT, WB 4101, RU 24969, 2-(4-[4-(2-pyrimidinyl)-1-piperazinyl] butyl)-1,2-benzisothiazol-3-(2H)one-1, 1-dioxidehydrochloride (TVX Q 7821), 5-methoxydimethyltryptamine, and d-lysergic acid diethylamide. The potencies of RU 24969, TFMPP, and quipazine for [3H]5-HT binding are increased by addition of 100 nM 8-OH-DPAT and 3,000 nM mianserin to the [3H]5-HT binding assay. Moreover, the drugs have apparent Hill slopes near 1 under these conditions. This subpopulation of total [3H]5-HT binding is designated 5-HT1B. By contrast, methysergide and mianserin become more potent inhibitors of residual [3H]5-HT binding to non-5-HT1A sites in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969. The drug competition curves under these conditions have apparent Hill slopes of near unity and these sites are designated 5-HT1C. Drug competition studies using a series of 24 agents reveals that each 5-HT1 subtype site has a unique pharmacological profile. These results suggest that radioligand studies can be used to differentiate three distinct subpopulations of 5-HT1 binding sites labeled by [3H]5-HT in rat frontal cortex.     

Elevated CO2 and herbivory influence trait integration in Arabidopsis thaliana

We lack information on how elevated CO₂, and its interaction with other factors like herbivory, affect levels and patterns of trait integration in plants. We experimentally tested the hypothesis that elevated CO₂ disrupts and restructures functional associations among plant traits, in the selfing annual, Arabidopsis thaliana. We tested for these effects both in the presence and absence of herbivory by larvae of the diamondback moth, Plutella xylostella. Elevated CO₂, both alone and combined with moth herbivory, modified integrated trait responses. In addition, integration under different environments was genotype‐specific. These results imply that global changes in CO₂ are likely to cause divergent evolutionary outcomes among populations of plants that differ in the initial structure of their quantitative genetic variation.     

E1/E2 type cation transport ATPases: Evidence for transient associations between protomers

E1/E2 type cation transport ATPases are known to exist in different conformeric states. Recent evidence characterizing these conformers in membrane is reviewed. A consensus view is proposed in which E2 conformers tend to form oligomeric complexes by lateral association between monomeric protomers and El conformers exhibit the opposite behaviour. It is suggested that transient associations between monomers during cation pump cycles may be a common feature of the ion translocation mechanism under physiological conditions.