Noriyuki Habuka

The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

BACKGROUND Hepatitis C virus (HCV) is the major etiological agent of hepatocellular carcinoma, and HCV RNA-dependent RNA polymerase (RdRp) is one of the main potential targets for anti-HCV agents. HCV RdRp performs run-off copying replication in an RNA-selective manner for the template-primer duplex and the substrate, but the structural basis of this reaction mechanism has still to be elucidated. RESULTS The three-dimensional structure of HCV RdRp was determined by X-ray crystallography at 2.5 A resolution. The compact HCV RdRp structure resembles a right hand, but has more complicated fingers and thumb domains than those of the other known polymerases, with a novel alpha-helix-rich subdomain (alpha fingers) as an addition to the fingers domain. The other fingers subdomain (beta fingers) is folded in the same manner as the fingers domain of human immunodeficiency virus (HIV) reverse transcriptase (RT), another RNA-dependent polymerase. The ribose-recognition site of HCV RdRp is constructed of hydrophilic residues, unlike those of DNA polymerases. The C-terminal region of HCV RdRp occupies the putative RNA-duplex-binding cleft. CONCLUSIONS The structural basis of the RNA selectivity of HCV RdRp was elucidated from its crystal structure. The putative substrate-binding site with a shallow hydrophilic cavity should have ribonucleoside triphosphate (rNTP) as the preferred substrate. We propose that the unique alpha fingers might represent a common structural discriminator of the template-primer duplex that distinguishes between RNA and DNA during the replication of positive single-stranded RNA by viral RdRps. The C-terminal region might exert a regulatory function on the initiation and activity of HCV RdRp.

The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNA-dependent RNA polymerase

We have previously determined the crystal structure of a non-structural 5B (NS5B) protein, an RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). NS5B protein with the hydrophobic C-terminal 21 amino acid residues truncated, designated NS5B(570), shows a typical nucleotide polymerase structure resembling a right-hand shape. In the crystal structure, a C-terminal region between Leu545 and His562 occupies a putative RNA-binding cleft of this polymerase and seems to inhibit the polymerase activity. Varieties of recombinant NS5B proteins (NS5B(552), NS5B(544), NS5B(536) or NS5B(531), with C-terminal 39, 47, 55 or 60 amino acid residues truncated, respectively) were systematically constructed to elucidate effects of the region on the polymerase activity. NS5B(544), NS5B(536) and NS5B(531) showed markedly higher RdRp activities compared to the activities of NS5B(570) or NS5B(552). Furthermore, when the hydrophobic amino acid residues Leu547, Trp550 and Phe551 (LWF) in NS5B(570) and NS5B(552) were changed to alanine, their activities were higher than that of the original NS5B(570). The crystal structures of the various recombinant NS5B proteins were also determined. Structural comparison of the NS5B proteins indicates that the activation was caused by elimination of a unique hydrophobic interaction between the three C-terminal residues and a shallowly concave pocket consisting of thumb and palm domains.

Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)+ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type α genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH2 and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type α genomic clone is a functional protein exhibiting RNA N-glycosidase activity.

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.

Escherichia coli ribosome is inactivated by Mirabilis antiviral protein which cleaves the N-glycosidic bond at A2660 of 23 S ribosomal RNA.

Ribosome-inactivating proteins (RIPs) are known to inactivate eukaryotic ribosomes, which results in the inhibition of protein synthesis, but there has been no evidence that they inactivate the ribosomes of Escherichia coli. Recently, Mirabilis antiviral protein (MAP), a RIP, has been shown to inhibit the protein synthesis of E. coli as well as eukaryotes. To elucidate its mechanism, E. coli ribosomes treated with MAP were analyzed by polyacrylamide/agarose composite gel electrophoresis and RNA sequencing using reverse transcriptase with DNA primer. The 23 S rRNAs, with an A260 value for ribosomes of 15, were completely cleaved in vitro by a 30 minute treatment with MAP at a concentration of 100 nM at 37 degrees C and a subsequent treatment with aniline. However, they were not affected by ricin A-chain under the same conditions. The primer extension of DNA polymerization stopped before A2660 of 23 S rRNA in RNA sequencing. Furthermore, both 16 S and 23 S rRNAs were cleaved by the MAP and aniline treatments when naked E. coli rRNAs were used as substrates, and the primer extension stopped before bases A2660 and A1014, respectively, in RNA sequencing. As the A2660 region has been shown to interact with the elongation factors EF-Tu and EF-G these results indicate that MAP cleaves the N-glycosidic bond at A2660 in E. coli 23 S RNA resulting in the inactivation of the ribosome.

Nucleotide sequence of a genomic gene encoding tritin, a ribosome-inactivating protein from Triticum aestivum.

A genomic gene of tritin, a ribosome-inactivating protein (RIP) from Triticum aestivum, was cloned using a barley RIP gene as a probe. The 5′-non-coding region has potential TATA boxes and three sequences homologous to the binding sequence of the transcriptional activator protein Opaque-2 which activates maize RIP gene expression. The cloned DNA encoded tritin consits of 275 amino acids with no secretion signal sequence. The coding region of tritin was expressed in Escherichia coli using lac promoter and yielded a protein similar to the native one, as determined by SDS-polyacrulamide gel electrophoresis and immunological analysis.

Expression of a pokeweed antiviral protein in Escherichia coli and its characterization

Two expression vectors were constructed to produce a putative mature α‐pokeweed antiviral protein (α‐PAP) in Escherichia coli with its NH2‐ and COOH‐terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2‐terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N‐glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.

Improved Crystals of the Toxic Protein MAP by Protein Engineering Towards the Host Specificity

Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)21) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.

Mode of action of DNA topoisomerase I from Alcaligenes sp.

Abstract A DNA topoisomerase I was partially purified from cell homogenate of a Gram-negative bacterium Alcaligenes sp. using centrifugation, streptomycin treatment, and DEAE-cellulose and P-cellulose chromatographies. The enzyme efficiently catalyzed the removal of superhelical turns from a negatively twisted DNA, requiring Mg2+ or Ca2+ for this activity. A broad pH range was exhibited, with maximum activity at pH 8.5–10.0. DNA-relaxing activity was seen by agarose gel electrophoresis and electron microscope observation.