Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a ß-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the ß-bulge loop is required for supercoiling activity. It has previously been shown that the ß-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a ß-bulge loop as a minimal latch is reported. It is shown that the ß-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

Analysis of helicase domain mutations in the hepatitis E virus derived from patients with fulminant hepatic failure: effects on enzymatic activities and virus replication.

Fulminant hepatic failure (FHF) is the severe form of hepatitis E virus infection. Virus sequence analyses from severe cases have shown presence of unique and highly conserved mutations in the helicase domain of genotype 1, 3 and 4 viruses. We evaluated role of two amino acid replacements (L1110F) and (V1120I); found to be frequent in genotype 1 FHF-E viruses from India. Three mutant helicase proteins (two with single point mutations and one with dual mutations) were expressed in Escherichia coli and evaluated for their ATPase and RNA unwinding activities. Both L1110F and V1120I helicase mutants showed marginal decrease in ATPase activity, while L1110F/V1120I dual mutant showed normal ATPase activity. All three mutants proteins showed RNA unwinding activities comparable to wild type protein. Corresponding mutations were made in the helicase domain of HEV RLuc replicon and replication efficiencies were tested in the S10-3 (Huh 7) cells. The mutant replicon V1120I showed lower replication as compared to L1110F and L1110F/V1120I mutants. However, all three replicon mutants showed lower replication efficiencies as compared to the wild type replicon. Walker A and Walker B motif mutant HEV replicons were unable to replicate indicating essential role of the virus encoded helicase domain during HEV replication. FHF-E associated helicase mutations resulted in only marginal decrease in the virus replication suggesting alternate function/s of the helicase protein. Mutations in the helicase domain of FHF-E viruses may be responsible for changing virus or host-virus protein-protein interactions, causing alterations in the host responses, eventually leading to more severe disease manifestations.

Mutagenesis of hepatitis E virus helicase motifs: effects on enzyme activity.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. The open reading frame 1 encoded non-structural polyprotein has putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase, however processing of this polyprotein is still uncertain. HEV helicase belongs to superfamily 1 and has all seven conserved motifs typical of the family. NTPase and RNA duplex unwinding activities of HEV helicase domain were recently demonstrated by us. A non-radioactive RNA unwinding assay was developed using biotin and digoxigenin labeled duplex RNA substrate with 5' overhangs for measuring strand displacement activity of the helicase. A series of deletion mutants were constructed to investigate role of individual motifs in the enzymatic activities. Deletion mutants for motif M I and M IV showed increase in ATPase activity. Deletion mutant M VI retained ATPase activity comparable to wild type protein. Mutant M II showed reduced ATPase activity (P=0.003) with no significant decrease in unwinding activity while mutants M Ia and M III showed major reduction of both ATPase and unwinding activities indicating crucial role of these motifs in the helicase function. Overall analysis of deletion mutants showed that Motif I, IV, V and VI have alternative motifs to carry out enzymatic functions of the protein while motifs Ia and III are critical as well as unique motifs in the protein. Knowing the important role of helicase protein during positive sense RNA virus replication, these unique motifs could be good antiviral targets.

An efficient synthesis of benzodiazepinyl phosphonates as clostripain inhibitors via FeCl3 catalyzed four-component reaction.

A novel one-pot route for the synthesis of benzodiazepinyl phosphonates (BDPs) has been achieved. FeCl(3) efficiently catalyzed four-component condensation of diamines, acetone and phosphites in the presence of molecular sieves to furnish BDPs as novel chemical entities with good yield. The synthesized BDPs have shown significant protease inhibition activity against clostripain, a disease model for gas gangrene, suggesting that these novel chemical entities could be further explored as cysteine protease inhibitors.