Expanded turn conformations: characterization and sequence-structure correspondence in alpha-turns with implications in helix folding

Like the beta-turns, which are characterized by a limiting distance between residues two positions apart (i, i+3), a distance criterion (involving residues at positions i and i+4) is used here to identify alpha-turns from a database of known protein structures. At least 15 classes of alpha-turns have been enumerated based on the location in the phi,psi space of the three central residues (i+1 to i+3)-one of the major being the class AAA, where the residues occupy the conventional helical backbone torsion angles. However, moving towards the C-terminal end of the turn, there is a shift in the phi,psi angles towards more negative phi, such that the electrostatic repulsion between two consecutive carbonyl oxygen atoms is reduced. Except for the last position (i+4), there is not much similarity in residue composition at different positions of hydrogen and non-hydrogen bonded AAA turns. The presence or absence of Pro at i+1 position of alpha- and beta-turns has a bearing on whether the turn is hydrogen-bonded or without a hydrogen bond. In the tertiary structure, alpha-turns are more likely to be found in beta-hairpin loops. The residue composition at the beginning of the hydrogen bonded AAA alpha-turn has similarity with type I beta-turn and N-terminal positions of helices, but the last position matches with the C-terminal capping position of helices, suggesting that the existence of a "helix cap signal" at i+4 position prevents alpha-turns from growing into helices. Our results also provide new insights into alpha-helix nucleation and folding.     

Evolutionary link between the mycobacterial plasmid pAL5000 replication protein RepB and the extracytoplasmic function family of σ factors

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10(-3)) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) σ factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of σ factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.     

Sulfated homologues of heparin inhibit hepatitis C virus entry into mammalian cells

The mechanism of entry of hepatitis C virus (HCV) through interactions between the envelope glycoproteins and specific cell surface receptors remains unclear at this time. We have previously shown with the vesicular stomatitis virus (VSV)/HCV pseudotype model that the hypervariable region 1 of the HCV E2 envelope glycoprotein helps in binding with glycosaminoglycans present on the cell surface. In this study, we have examined the binding of HCV envelope glycoproteins with chemically modified derivatives of heparin. Furthermore, we have determined the functional relevance of the interaction of heparin derivatives with HCV envelope glycoproteins for infectivity by using a human immunodeficiency virus (HIV)/HCV pseudotype, a VSV/HCV pseudotype, and cell culture-grown HCV genotype 1a. Taken together, our results suggest that the HCV envelope glycoproteins rely upon O-sulfated esters of a heparin homologue to facilitate entry into mammalian cells.     

Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain

A sialyltransferase involved in the biosynthesis in vitro of LD1c (NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-NAc beta 1-3Gal beta 1-4Glc-Cer) has been characterized from 9 to 11-day-old embryonic chicken brains. The CMP-[14C]NeuAc:LM1(alpha 2-8)sialyltransferase (SAT-2) sedimented (75%) at the junction of 0.75 and 1.2 M on a discontinuous sucrose density gradient when still membrane bound. In addition to the biosynthesis of LD1c, the detergent-solubilized (0.4% Nonidet P-40) preparation also catalyzes the transfer of sialic acid to O-8 of sialic acid in GM3 to form GD3 (NeuAc alpha 2-8NeuAc alpha 2 - 3Gal beta 1 - 4Glc - Cer). Substrate inhibition studies indicated that these two reactions are probably catalyzed by the same enzyme, SAT-2. The kinetic parameters of SAT-2 activity were determined. The Km values were 70 and 63 microM with CMP-[14C]NeuAc and LM1, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The (alpha 2-8)-linkage between the terminal and penultimate sialic acids was determined using nonradioactive CMP-NeuAc and [Ac-14C]LM1 as substrates (Higashi, H., and Basu, S. (1982) Anal. Biochem. 120, 159-164) for the enzyme, followed by identification of the permethylated [14C]sialic acid of the product by radioautography. At 0.5 mM N-ethylmaleimide, the SAT-2 activity was inhibited 50% whereas SAT-1 and SAT-3 activities (Basu, M., Basu, S., Stoffyn, A., and Stoffyn, P. (1982) J. Biol. Chem. 257, 12765-12769) remained uninhibited.     

Inhibition of DNA replication fork progression and mutagenic potential of 1, N6-ethenoadenine and 8-oxoguanine in human cell extracts

Comparative mutagenesis of 1,N⁶-ethenoadenine (εA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past εA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, εA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G→T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of εA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the εA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, εA→T mutations increased 3-fold in products derived from the mismatched εA: G construct compared with those derived from the εA: T construct. More efficient repair of εA in the εA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol η have shown that this polymerase is highly error-prone when bypassing εA. To examine if pol η is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The εA: T construct induced εA→G and εA→C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, εA→T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol η-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA.     

Effects of prostaglandins E2 and F2α on lecithin biosynthesis by cultured lung cells

Transformed cells from human lung carcinoma (Line A549), resembling type II pneumocytes, were cultured in monolayer at 37°C and incubated for five hours with ³H-choline and ¹⁴C-palmitate in the presence of various concentrations of prostaglandins (PGs) E₂ and F_2α. In the control (no PG) the level of % palmitate incorporation was 13.5 × as high as that of choline, after taking isotope dilution into account. Between the concentrations studied, 0.1 and 10 μM, both prostaglandins stimulated markedly the incorporation of both precursors, though choline up to 3 × better than palmitate. This was indicated by a change in the palmitate/choline incorporation ratio from 13.5 to as low as 4.2. At the lowest PG concentration, 0.1 μM, PGE₂ was much more effective than PGF_2α in stimulating the incorporation of both precursors.     

Human brain creatine kinase binding to immobilized cibacron blue F3GA: characterization and use in purification of the enzyme.

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) from adult human brain grey matter was purified by cibacron blue F3GA-Sepharose affinity chromatography. By gel electrophoresis of the purified enzyme under non-denaturing conditions a single protein band was observed. The dye-bound enzyme was eluted using its substrate, ATP. Reversibility of the binding of purified creatine kinase to blue Sepharose by ATP in a concentration-dependent manner indicated that the cibacron blue molecule which structurally mimics nucleotides occupied the substrate binding site of the enzyme. Also the marked dependence of enzyme binding to blue Sepharose on Mg2+ concentration suggested that Mg2+ ion is capable of combining with the dye moiety to form a site-specific binding complex that is similar to the physiological substrate of creatine kinase, namely Mg(2+)-ATP or Mg(2+)-ADP.     

Activation of human spleen glucocerebrosidases by monoacylglycol sulfates and diacylglycerol sulfates

The study of the acidic lipid requirement of human spleen glucocerebrosidase was extended to include two new series of acidic lipids, namely, monoacylglycol sulfates and diacylglycerol sulfates. Lysosomal glucocerebrosidase was extracted with sodium cholate and 1-butanol to render its beta-glucosidase activity dependent upon exogenous lipids. Maximum reactivation of control glucocerebrosidase was obtained with nonanoylglycol sulfate (NGS) and diheptanoylglycerol sulfate (DHGS). However, the effects of these lipids were markedly dependent on the nature of buffer used in the assay medium; specifically, 0.2 M sodium citrate-phosphate (pH 5.5) was much more effective than 0.2 M sodium acetate (pH 5.5) in permitting these lipids to reactivate glucocerebrosidase. In contrast, the marked activation of glucocerebrosidase by phosphatidylserine and galactocerebroside 3-sulfate (sulfatide) that was achievable in the sodium acetate buffer was totally inhibited by citrate or phosphate ions. The effects of NGS and DHGS on the kinetic parameters of control glucocerebrosidase were to lower the Km for the substrate, 4-methylumbelliferyl-beta-D-glucoside from 5.5 mM to approximately 2 mM (in sodium citrate-phosphate buffer) and markedly increase the Vmax. Furthermore, with DHGS, significant activation was achieved at concentrations below the lipid's critical micellar concentration. None of the monoacylglycol- or diacylglycerol sulfates were capable of stimulating mutant glucocerebrosidases from either type 1 (Ashkenazi-Jewish) or type 2 Gaucher's disease patients. Like control glucocerebrosidase, the type 1 glucocerebrosidase was unresponsive to phosphatidylserine and sulfatide when the beta-glucosidase assay was conducted in 0.2 M sodium citrate-phosphate buffer. Based on the differential action of these lipid activators in the two buffers and their effects on the mutant enzymes, we propose that, with regard to the lipid requirement of glucocerebrosidase, there are two classes of acidic lipids--one comprised of phosphatidylserine and sulfatide and the other comprised of the likes of NGS, DHGS, or sodium taurodeoxycholate. It appears that control glucocerebrosidase and the mutant enzyme of the patient with type 1 Gaucher's disease is reconstitutable with the first class of lipids whereas the glucocerebrosidase of the type 2 patient is not. The observations in this report are interpreted in terms of a model which postulates that normal glucocerebrosidase possesses at least two distinct lipid binding domains.