The crystal structure of the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) has been determined at a resolution of 2.4 A and refined to a crystallographic R factor of 0.20. The protein folds into a five-stranded mixed beta sheet flanked by an asymmetric distribution of four alpha helices. Two divalent metal cations bind in the active site surrounded by a cluster of four conserved acidic amino acid residues. The overall structure is similar in most respects to the RNase H from Escherichia coli. Structural features characteristic of the retroviral protein suggest how it may interface with the DNA polymerase domain of p66 in the mature RT heterodimer. These features also offer insights into why the isolated RNase H domain is catalytically inactive but when combined in vitro with the isolated p51 domain of RT RNase H activity can be reconstituted. Surprisingly, the peptide bond cleaved by HIV-1 protease near the polymerase-RNase H junction of p66 is completely inaccessible to solvent in the structure reported here. This suggests that the homodimeric p66-p66 precursor of mature RT is asymmetric with one of the two RNase H domains at least partially unfolded.

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.

The bifunctional enzyme dihydrofolate reductase-thymidylate synthase catalyses both the reductive methylation of 2'-deoxyuridylate and the subsequent reduction of dihydrofolate to yield 2'-deoxythymidylate and tetrahydrofolate at two spacially discrete sites situated on different protein domains. The X-ray structure of dihydrofolate reductase-thymidylate synthase from Leishmania major indicates that transfer of dihydrofolate between these sites does not occur by transient binding at both sites but rather by movement of dihydrofolate across the surface of the protein. The enzyme has an unusual surface charge distribution that could account for this channelling of dihydrofolate between active sites.

The three-dimensional structure of phosphoribosylglycinamide formyltransferase (10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2) has been solved both as an apoenzyme at 2.8-A resolution and as a ternary complex with the substrate glycinamide ribonucleotide and a folate inhibitor at 2.5-A resolution.The structure is a modified doubly wound a/S sheet with flexibility in the active site, including a disordered loop in the apo structure, which is ordered in the ternary complex structure. This enzyme is a target for anti-cancer therapy and now for structure-based drug design. (3) and is currently completing phase I clinical trials in a number of centers. This enzyme is, therefore, a good target for anti-cancer therapy and structure-based drug design. In prokaryotes, GART is found as a single protein but in most eukaryotes it is found as the C-terminal portion of a large multifunctional protein (Mr > 100,000) also containing GAR synthetase and aminoimidazole ribonucleotide synthetase activities. The sequences of GART from prokaryotes and eukaryotes are homologous (4, 5), and the structures are expected to be similar.Here we report the three-dimensional structure of Escherichia coli GART as an apoenzyme (2.8-A resolution) and also as a ternary complex (2.5-A resolution) with the substrate GAR and a folate-based inhibitor, 5-deaza-5,6,7,8-tetrahydrofolate (5dTHF) (Fig. 1). The GART structure is a modification of the classic doubly wound a/3 sheet with the active site located at the C-terminal edge, near the middle of the seven- stranded 83-sheet. We describe the major changes in the structure between the complexed and uncomplexed forms, interpret structure-activity relationships (SAR), and discuss the mechanism for catalysis based on our structural results.MATERIALS AND METHODS The coding region ofE. coli GART was amplified from E. coli K-12 chromosomal DNA with PCR method (6). Primer sequences were derived from E. coli purN gene (5). Amplified DNA was confirmed and placed downstream of the T7 bacteriophage gene 10 promoter (7) as the second cistron using modified E. coli DHFR translation initiation region as the first cistron (8,9). The E. coli strain AP401-harboring expression plasmid was induced as described (9).GART was purified by using a DEAE-Sephacel column elution with a 0.03-0.8 M NaCl gradient followed by a Sephadex G75 column and a fast protein liquid chromotography mono Q elution with a 0.03-1 M NaCl gradient. The specific activity of the purified enzyme was 10 Jumol/min per mg. All steps were done at 40C in 50 mM Tris, pH 7.5/30 mM NaCl/1 mM dithiothreitol. A similar purification has since been published (10).All crystals were grown at 20'C by hanging-drop vapor diffusion. Apo crystals grew from 30 mg of protein per ml in 50 mM Tris (pH 7.5), 1 mM dithiothreitol, and a reservoir of 0.8 M Na+,K+ phosphate (pH 6.75) similar to conditions in Abbreviations: GAR, glycinamide ribonucleotide; GART, phosphoribosylglycinamide formyltransferase; DHFR, dihydrofolate reduct...

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