Helicases are nucleotide triphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication. The 2.1 A resolution structure of the HCV helicase from the positive-stranded RNA hepatitis C virus reveals a molecule with distinct NTPase and RNA binding domains. The structure supports a mechanism of helicase activity involving initial recognition of the requisite 3' single-stranded region on the nucleic acid substrate by a conserved arginine-rich sequence on the RNA binding domain. Comparison of crystallographically independent molecules shows that rotation of the RNA binding domain involves conformational changes within a conserved TATPP sequence and untwisting of an extended antiparallel beta-sheet. Location of the TATPP sequence at the end of an NTPase domain beta-strand structurally homologous to the 'switch region' of many NTP-dependent enzymes offers the possibility that domain rotation is coupled to NTP hydrolysis in the helicase catalytic cycle.

The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.

Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson’s disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique beta-hairpin in the thumb subdomain which protrudes toward the active site. We report here that this beta-hairpin plays an important role in positioning the 3' terminus of the viral RNA genome for correct initiation of replication. The presence of this beta-hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsRNA) molecules and allows only the single-stranded 3' terminus of an RNA template to bind productively to the active site. We propose that this beta-hairpin may serve as a "gate" which prevents the 3' terminus of the template RNA from slipping through the active site and ensures initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a beta-hairpin deletion mutant that utilizes dsRNA substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may represent a novel replication strategy adopted by HCV and related viruses.

Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single-chain recombinant form of the protease has been constructed in which NS4A residues 21-32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3-181) through a tetrapeptide linker. The single-chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size-exclusion chromatography. The single-chain recombinant protease domain shows full proteolytic activity cleaving the NS5A-5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a K, and kc,, of 20.0 k 2.0 p M and 9.6 t 2.0 min-I, respectively; parameters identical to those of the authentic NS31-631/NS4Al~s4 protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392-3401).

The structure of the histidine-binding protein (HBP, M(r) = 26,100), involved solely in active transport, has been determined by the molecular replacement technique and refined to 1.89-A resolution and to an R-factor of 0.199. The structure is that of two protein molecules, each with a bound L-histidine, in the asymmetric unit. Replacement solution was achieved by using a model of the crystal structure of the ligand-free, open-cleft form of the lysine/arginine/ornithine-binding protein which was modified so that the two domains are close to each other by bending the hinge connecting the two domains. The bound histidine is held in place by 10 hydrogen bonds, 2 salt links, and about 60 van der Waals contacts. Elucidation of the HBP structure brings a total of eight different binding proteins structures determined in our laboratory, including those with specificities for monosaccharides, maltodextrins (linear and cyclic), aliphatic amino acids, and inorganic oxyanions. These structures comprise about a third of the entire family of periplasmic binding proteins which act as initial primary high-affinity receptors of active transport in Gram-negative bacteria. Two of the binding proteins with specificities for glucose/galactose and maltodextrins also serve in a similar capacity in chemotaxis. Though these proteins have different molecular weights (ranging from 26,000 to 40,000), amino acid sequences, and ligand specificities, their three-dimensional structures are similar overall. They are elongated (axial ratios of 2:1) and composed of two similar globular domains separated by a deep cleft wherein the ligand-binding site is located. These structures provide understanding of molecular recognition of a variety of ligands at the atomic level and functional roles of the binding proteins.

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Three helicase structures have been determined recently: that of the DNA helicase PcrA, that of the hepatitis C virus RNA helicase, and that of the Escherichia coli DNA helicase Rep. PcrA and Rep belong to the same super-family of helicases (SFI) and are structurally very similar. In contrast, the HCV helicase belongs to a different super-family of helicases, SF2, and shows little sequence homology with the PcrA/Rep helicases. Yet, the HCV helicase is structurally similar to Rep/PcrA, suggesting preservation of structural scaffolds and relationships between helicase motifs across these two super-families. The comparison study presented here also reveals the existence of a new helicase motif in the SF1 family of helicases.Keywords: HCV, helicase; helicase motif; Rep; structure Helicases are enzymes that unwind duplex DNA or RNA in reactions coupled to nucleoside 5' triphosphate (NTP) binding and hydrolysis, and play central roles in processes affecting nucleic acids (Lohman & Bjomson, 1996). On the basis of amino acid sequence homology, helicases have been classified into five superfamilies (Gorbalenya & Koonin, 1993). Although the overall sequence homology is low, conserved regions or "helicase" motifs within each family have been identified (Gorbalenya et al., 1989). However, homology across families is very weak and limited to the signature sequences that are required for NTP binding (helicase motifs I and 11) (Koonin & Dolja, 1993). With the exception of these NTP binding sequences, it has not been clear prior to the structural comparisons reported here whether the other motifs share common functional roles.Recently, structural information on three helicases has been reported at atomic resolution. The first view of a DNA-helicase, that of the PcrA helicase from Bacillus stearothemzophilus, revealed a two-domain structure (domains 1 and 2), with each domain consisting of two subdomains (lA, lB, 2A, and 2B) (Subramanya et al., 1996). While subdomains 1B and 2B are mostly a-helical and are formed by large insertions within subdomains 1A and 2A (Fig. lA) (Fig. IA). The second view of a helicase was that of the RNA helicase domain of the NS3 protein of hepatitis C virus (HCV) (Yao et al., 1997) (Fig. 1B). The HCV helicase consists of three distinct domains, two of which are RecA structural homologues. The third domain is mostly a-helical, and is formed by the C-terminal region of the sequence. Finally, a third view of a DNA helicase structure was recently obtained: that of the Rep DNA helicase bound to single-stranded DNA (ss-DNA) (Korolev et al., 1997). The asymmetric unit of the Rep/ss-DNA crystals contained two molecules that differ markedly by a large reorientation of the subdomain structure. Although one of the molecules (the open conformer) is very similar in structure to the PcrA DNA-helicase (Fig. lA), the other molecule (the closed form) has undergone a swiveling motion of its 2B domain, corresponding to a 130" rotation around the hinge region that connects domains 2A and 2B.In an attempt to deter...