Purification and characterization of the N-terminal nucleotide binding domain of an ABC drug transporter of Candida albicans: uncommon cysteine 193 of Walker A is critical for ATP hydrolysis

The Candida drug resistance protein Cdr1p (approximately 170 kDa) is a member of ATP binding cassette (ABC) superfamily of drug transporters, characterized by the presence of 2 nucleotide binding domains (NBD) and 12 transmembrane segments (TMS). NBDs of these transporters are the hub of ATP hydrolysis activity, and their sequence contains a conserved Walker A motif (GxxGxGKS/T). Mutations of the lysine residue within this motif abrogate the ability of NBDs to hydrolyze ATP. Interestingly, the sequence alignments of Cdr1p NBDs with other bacterial and eukaryotic transporters reveal that its N-terminal NBD contains an unusual Walker A sequence (GRPGAGCST), as the invariant lysine is replaced by a cysteine. In an attempt to understand the significance of this uncommon positioning of cysteine within the Walker A motif, we for the first time have purified and characterized the N-terminal NBD (encompassing first N-terminal 512 amino acids) of Cdr1p as well as its C193A mutant protein. The purified NBD-512 protein could exist as an independent functional general ribonucleoside triphosphatase with strong divalent cation dependence. It exhibited ATPase activity with an apparent K(m) in the 0.8-1.0 mM range and V(max) in the range of 147-160 nmol min(-)(1) (mg of protein)(-)(1). NBD-512-associated ATPase activity was also sensitive to inhibitors such as vanadate, azide, and NEM. The Mut-NBD-512 protein (C193A) showed a severe impairment in its ability to hydrolyze ATP (95%); however, no significant effect on ATP (TNP-ATP) binding was observed. Our results show that C193 is critical for N-terminal NBD-mediated ATP hydrolysis and represents a unique feature distinguishing the ATP-dependent functionality of the ABC transporters of fungi from those found in bacteria and other eukaryotes.     

Efficacy of alkaloid (-)-corypalmine against spore germination of some fungi

Inhibition activity of the alkaloid (−)-corypalmine on spore germination of plant pathogenic and saprophytic fungi (Alternaria solani, A brassicicola, A. brassicae, A. melongenae, Curvularia pallescens, C. lunata, C. maculans, Curvularia sp.,Colletotrichum sp.,Helminthosporium speciferum, H. frumentacei, H. pennisetti, Heterosporium sp.,Penicillum sp.,Ustilago cynodontis) was determined. Spore germination of all the tested fungi was inhibited,Heterosporium sp. andUstilago cynodontis being the most sensitive (complete inhibition of spore germination was observed at the very low concentration of 200 ppm).Curvularia palliscens, C. maculans andCurvularia sp. were less sensitive; complete inhibition of spore germination occurred at 400 ppm.     

pH and cation-induced thermodynamic stability of human hyaluronan binding protein 1 regulates its hyaluronan affinity

Hyaluronan-binding protein 1 (HABP1) is a trimeric protein with high negative charges distributed asymmetrically along the faces of the molecule. Recently, we have reported that HABP1 exhibits a high degree of structural flexibility, which can be perturbed by ions under in vitro conditions near physiological pH (Jha, B. K., Salunke, D. M., and Datta, K. (2003) J. Biol. Chem. 278, 27464-27472). Here, we report the effect of ionic strength and pH on thermodynamic stability of HABP1. Trimeric HABP1 was shown to unfold reversibly upon dissociation ruling out the possibility of existence of folded monomer. An increase in ionic concentration (0.05-1 M) or decrease in pH (pH 8.0-pH 5.0) induced an unusually high thermodynamic stability of HABP1 as reflected in the gradual increase in transition midpoint temperature, enthalpy of transition, and conformational entropy. Our studies suggest that the presence of counter ions in the molecular environment of HABP1 leads to dramatic reduction of the intramolecular electrostatic repulsion either by de-ionizing the charged amino acid residues or by direct binding leading to a more stable conformation. A regulation on cellular HA-HABP1 interaction by changes in pH and ionic strength may exist, because the more stable conformation attained at higher ionic strength or at acidic pH showed maximum affinity toward HA as probed either in solid phase binding assay on HA-immobilized plates or an in-solution binding assay using intrinsic fluorescence of HABP1.     

Dual engagement regulation of protein interactions with the AP-2 adaptor alpha appendage

Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded alpha subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NECAP1, binds a second interaction site on the bilobal alpha appendage, located on the N-terminal beta sandwich subdomain. Both alpha appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the alpha appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.     

Circadian clocks and life-history related traits: Is pupation height affected by circadian organization in<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>?

InD. melanogaster, the observation of greater pupation height under constant darkness than under constant light has been explained by the hypothesis that light has an inhibitory effect on larval wandering behaviour, preventing larvae from crawling higher up the walls of culture vials prior to pupation. If this is the only role of light in affecting pupation height, then various light : dark regimes would be predicted to yield pupation heights intermediate between those seen in constant light and constant darkness. We tested this hypothesis by measuring pupation height under various light : dark regimes in four laboratory populations ofDrosophila melanogaster. Pupation height was the greatest in constant darkness, intermediate in constant light, and the least in a light/dark regime of LD 14:14 h. The results clearly suggest that there is more to light regime effects on pupation height than mere behavioural inhibition of wandering larvae, and that circadian organization may play some role in determining pupation height, although the details of this role are not yet clear. We briefly discuss these results in the context of the possible involvement of circadian clocks in life-history evolution.     

Arsenic exposure alters activity behaviour of key nitrogen assimilatory enzymes in growing rice plants

Rice seedlings when grown in sand cultures for 5–20 days under 25 and 50 M As₂O₃ in the medium showed a marked decline in growth when compared to controls. Increased absorption of arsenic from the medium, against the concentration gradient was observed. Greater localization of absorbed arsenic was noted in roots than in shoots. Rice plants grown for 20 days with 50 mol l^–1 arsenic in the medium accumulated upto 370 mol arsenic kg^–1 dry weight in roots. Increasing levels of As₂O₃ in situ caused a marked decline in the activities of the nitrate assimilatory enzymes nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS), whereas an increase in the activities of alanine and aspartate aminotransferases was observed. The activities of aminating (NADH-GDH) and deaminating (NAD⁺-GDH) glutamate dehydrogenases increased at moderately toxic level (25 M) of As₂O₃ whereas a higher As level of 50 M was inhibitory to the enzymes. Addition of 1 M proline in the reaction medium caused significant restoration in As-led loss of NR and GS activities. NR and GS extracted from arsenic exposed seedlings showed higher K _m values compared to the enzymes extracted from control-grown seedlings, whereas GDHs extracted from As-stressed seedlings showed a decrease in K _m. Results suggest that inhibition in the activities of N assimilatory enzymes accompanied with decreased affinity of the enzymes towards their substrates would eventually lead to a marked suppression of N assimilation and impaired growth of rice seedlings in As polluted environment.     

Sequence analysis of the long arm of rice chromosome 11 for rice–wheat synteny

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10× redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.     

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.     

Quantitative structure–activity relationship study on some benzodiazepine derivatives as anti-Alzheimer agents

A QSAR study was performed in an attempt to explore the pharmacophore of some benzodiazepine derivatives as anti-Alzheimer agents for the inhibition of -secretase. The study, which used the electrotopological state atom (ETSA) index, which encodes electronic and topological information, reveals the importance of two phenyl rings—one substituted and another unsubstituted, for the inhibition of the enzyme. Fluorine substitution on the substituted phenyl ring has an important contribution to the activity. R-configurations of the aliphatic chain substituents provide the exact conformation of the molecules to enter into the binding pockets of the receptor(s).Figure General structure of benzodiazepine containing -secretase inhibitors