NMR structure of the viral peptide linked to the genome (VPg) of poliovirus

VPgs are essential for replication of picornaviruses, which cause diseases such as poliomyelitis, foot and mouth disease, and the common cold. VPg in infected cells is covalently linked to the 5' end of the viral RNA, or, in a uridylylated form, free in the cytoplasm. We show here the first solution structure for a picornaviral VPg, that of the 22-residue peptide from poliovirus serotype 1. VPg in buffer is inherently flexible, but a single conformer was obtained by adding trimethylamine N-oxide (TMAO). TMAO had only minor effects on the TOCSY spectrum. However, it increased the amount of structured peptide, as indicated by more peaks in the NOESY spectrum and an up to 300% increase in the ratio of normalized NOE cross peak intensities to that in buffer. The data for VPg in TMAO yielded a well defined structure bundle with 0.6 A RMSD (versus 6.6 A in buffer alone), with 10-30 unambiguous constraints per residue. The structure consists of a large loop region from residues 1 to 14, from which the reactive tyrosinate projects outward, and a C-terminal helix from residues 18 to 21 that aligns the sidechains of conserved residues on one face. The structure has a stable docking position at an area on the poliovirus polymerase crystal structure identified as a VPg binding site by mutagenesis studies. Further, UTP and ATP dock in a base-specific manner to the reactive face of VPg, held in place by residues conserved in all picornavirus VPgs.     

Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif.

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.     

Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.Members of the Picornaviridae family of small RNA viruses cause a wide range of diseases in humans, including liver disease, heart disease, aseptic meningitis, the common cold, and poliomyelitis. The picornaviruses include the most common human viruses, which are the rhinoviruses that spread through respiratory pathways, and the second most common viruses, which are enteroviruses that spread by fecal-oral transmission. These viruses have ≈7.5-kb positive-sense genomes containing a single large open reading frame encoding a ≈250-kDa polyprotein that is cleaved into about a dozen different proteins by viral proteases (20). Their genome life cycle is completely RNA based, with replication being driven by the viral 3D^pol protein, an RNA-dependent RNA polymerase (RdRP).After viral RNA translation and polyprotein processing, 3D^pol replicates the infecting positive-strand RNA template into a negative-strand intermediate that is subsequently used as a template for positive-strand synthesis. During these processes, 3D^pol interacts with multiple templates, substrates, and other viral proteins; however, many aspects of these events remain obscure. The crystal structures of several picornaviral 3D^pol enzymes have been solved, and these all conform to the “right hand” analogy commonly used to describe polymerases as having palm, thumb, and finger domains (10, 14, 18, 22, 27, 29). Based on homology to other polymerases and the structures of 3D^pol-RNA complexes with foot-and-mouth disease and Norwalk virus polymerases, the finger domain interacts with the template RNA, the palm domain contains the active-site aspartate residues that coordinate the metals needed for catalysis, and the thumb domain contacts the exiting duplex RNA product (13, 26, 30).Poliovirus is among the most-studied picornaviruses, and its polymerase has been thoroughly characterized biochemically (3, 15) and structurally (28, 29). Processive RNA synthesis requires the formation of a stable 3D^pol elongation complex through a multistep process involving at least two conformational changes (2). First, following RNA binding, there is a slow (t_1/2 ≈ 12 s) conformational change that results in a 3D^pol-RNA complex poised for nucleoside triphosphate (NTP) incorporation. Second, following the addition of the first nucleotide to the primer, there is another conformational change to produce a very stable elongation complex with an in vitro half-life on the order of several hours. The polymerase begins processive elongation after the formation of this stable elongation complex, and each nucleotide addition cycle involves a five-step mechanism, of which NTP repositioning and NTP catalysis are rate limiting (3). Similar experiments using the homologous foot-and-mouth disease virus polymerase reveal an analogous set of complexes resulting in an elongation complex with a half-life of 27 h (1). Although these viral polymerase complexes have been well characterized biochemically, there is relatively little known about any structural changes involved in elongation complex formation or the catalytic cycle itself; all the 3D^pol structures solved thus far show essentially the same conformation, with no evidence for significant conformational changes upon RNA or NTP binding.The activation of several picornaviral polymerases is dependent upon correct cleavage of 3D^pol from the viral 3CD^pro precursor protein in order to create a new N terminus that can be buried in a pocket at the base of the 3D^pol finger domain. This buried N terminus has been observed in poliovirus, coxsackievirus, rhinovirus, and foot-and-mouth disease virus polymerases (10, 14, 22, 29). In solving the structure of poliovirus polymerase, we observed that burying the N terminus resulted in a subtle but important conformational change in the active site whereby Asp238 was repositioned to make a key hydrogen bond with the 2′ hydroxyl of a bound NTP (29). Addition or deletion of a single residue at the N terminus abolished enzyme activity, and mutation of Gly1 to alanine resulted in a partially active enzyme with slightly altered positioning of Asp238. Further data from coxsackievirus polymerase showed that the addition of a second N-terminal glycine also inactivated the enzyme, but activity could be restored by also deleting Glu2, indicating that there is a specific length requirement in the N-terminal sequence of the enzyme (10). A prime candidate for involvement in such a length requirement is residue Phe5 of coxsackievirus 3D^pol that corresponds to Trp5 in poliovirus 3D^pol. In the 3D^pol structures, there is a backbone distortion in the β-strand composed of residues 1 to 9 that results in this large hydrophobic amino acid being solvent exposed rather than buried in an adjacent hydrophobic pocket (Fig. ​(Fig.11 A). This unusual conformation at residue 5 is conserved among picornaviral polymerase structures, and substitution mutations at this residue had significant effects on coxsackievirus polymerase activity (10). Large hydrophobic amino acids at residue 5 increased 3D^pol activity, while small amino acids at residue 5 decreased 3D^pol activity (10). Based on these data, we previously proposed that the 3D^pol catalytic cycle involves a conformational change wherein residue 5 flips into an adjacent hydrophobic patch on the polymerase to aid in NTP positioning, and such a conformational change would require the N terminus to be correctly buried to act as a stable pivot for the rotational movement.Open in a separate windowFIG. 1.Elongation complex formation. (A) Structure of poliovirus polymerase showing the distortion of the β-sheet conformation between residues 3 and 4 that results in Trp5 being solvent exposed adjacent to a large hydrophobic patch composed of residues from the index (green) and middle (orange) fingers. (B) Cartoon of the PETE (polymerase elongation template element) RNAs used in complex formation assays. Both RNAs are G-less until the sixth nucleotide from the end, which limits secondary structure and stops elongation before the 5′ end to avoid possible end effects. The asterisk indicates the position of the amino-modified deoxythymidine where the IRDye label is covalently attached. (C) Denaturing PAGE showing the time course for formation of the +1 and +2 products from the two RNAs. Elongation complexes were formed by incubating 1 μM each PETE RNA, 15 μM 3D^pol, and 40 μM ATP and GTP at 22°C for various times as indicated. (D) Kinetics of +2 complex formation rates obtained from band intensity data curve fit to a single exponential. The resulting formation time constants are listed in Table ​Table11.In this work, we have investigated the role of residue 5 in 3D^pol in further detail by examining how a series of mutations in poliovirus 3D^pol affects RNA binding, elongation complex formation, elongation rate, and elongation complex stability. The data show that residue 5 mutations have major effects on the stability of the elongation complex, with minor effects on elongation complex formation and no effect on RNA binding affinities and elongation rates. Replication defects are also observed in the context of viral replication centers where residue 5 mutations significantly reduce RNA synthesis in cell-free coupled translation-replication reactions and slow the growth of infectious virus in cells.     

Foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe

The nucleoside analogue ribavirin (R) is mutagenic for foot-and-mouth disease virus (FMDV). Passage of FMDV in the presence of increasing concentrations of R resulted in the selection of FMDV with the amino acid substitution M296I in the viral polymerase (3D). Measurements of progeny production and viral fitness with chimeric viruses in the presence and absence of R documented that the 3D substitution M296I conferred on FMDV a selective replicative advantage in the presence of R but not in the absence of R. In polymerization assays, a purified mutant polymerase with I296 showed a decreased capacity to use ribavirin triphosphate as a substrate in the place of GTP and ATP, compared with the wild-type enzyme. The results suggest that M296I has been selected because it attenuates the mutagenic activity of R with FMDV. Replacement M296I is located within a highly conserved stretch in picornaviral polymerases which includes residues that interact with the template-primer complex and probably also with the incoming nucleotide, according to the three-dimensional structure of FMDV 3D. Given that a 3D substitution, distant from M296I, was associated with resistance to R in poliovirus, the results indicate that picornaviral polymerases include different domains that can alter the interaction of the enzyme with mutagenic nucleoside analogues. Implications for lethal mutagenesis are discussed.     

Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase

The active RNA-dependent RNA polymerase of poliovirus, 3Dpol, is generated by cleavage of the 3CDpro precursor protein, a protease that has no polymerase activity despite containing the entire polymerase domain. By intentionally disrupting a known and persistent crystal packing interaction, we have crystallized the poliovirus polymerase in a new space group and solved the complete structure of the protein at 2.0 A resolution. It shows that the N-terminus of fully processed 3Dpol is buried in a surface pocket where it makes hydrogen bonds that act to position Asp238 in the active site. Asp238 is an essential residue that selects for the 2' OH group of substrate rNTPs, as shown by a 2.35 A structure of a 3Dpol-GTP complex. Mutational, biochemical, and structural data further demonstrate that 3Dpol activity is exquisitely sensitive to mutations at the N-terminus. This sensitivity is the result of allosteric effects where the structure around the buried N-terminus directly affects the positioning of Asp238 in the active site.     

Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase.

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.     

Structure of a bifunctional DNA primase-polymerase

Genome replication generally requires primases, which synthesize an initial oligonucleotide primer, and DNA polymerases, which elongate the primer. Primase and DNA polymerase activities are combined, however, in newly identified replicases from archaeal plasmids, such as pRN1 from Sulfolobus islandicus. Here we present a structure-function analysis of the pRN1 primase-polymerase (prim-pol) domain. The crystal structure shows a central depression lined by conserved residues. Mutations on one side of the depression reduce DNA affinity. On the opposite side of the depression cluster three acidic residues and a histidine, which are required for primase and DNA polymerase activity. One acidic residue binds a manganese ion, suggestive of a metal-dependent catalytic mechanism. The structure does not show any similarity to DNA polymerases, but is distantly related to archaeal and eukaryotic primases, with corresponding active-site residues. We propose that archaeal and eukaryotic primases and the prim-pol domain have a common evolutionary ancestor, a bifunctional replicase for small DNA genomes.     

Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.     

Identification of the oriI-binding site of poliovirus 3C protein by nuclear magnetic resonance spectroscopy

Replication of picornaviral genomes requires recognition of at least three cis-acting replication elements: oriL, oriI, and oriR. Although these elements lack an obvious consensus sequence or structure, they are all recognized by the virus-encoded 3C protein. We have studied the poliovirus 3C-oriI interaction in order to begin to decipher the code of RNA recognition by picornaviral 3C proteins. oriI is a stem-loop structure that serves as the template for uridylylation of the peptide primer VPg by the viral RNA-dependent RNA polymerase. In this report, we have used nuclear magnetic resonance (NMR) techniques to study 3C alone and in complex with two single-stranded RNA oligonucleotides derived from the oriI stem. The (1)H-(15)N spectra of 3C recorded in the presence of these RNAs revealed site-specific chemical shift perturbations. Residues that exhibit significant perturbations are primarily localized in the amino terminus and in a highly conserved loop between residues 81 and 89. In general, the RNA-binding site defined in this study is consistent with predictions based on biochemical and mutagenesis studies. Although some residues implicated in RNA binding by previous studies are perturbed in the 3C-RNA complex reported here, many are unique. These studies provide unique site-specific insight into residues of 3C that interact with RNA and set the stage for detailed structural investigation of the 3C-RNA complex by NMR. Interpretation of our results in the context of an intact oriI provides insight into the architecture of the picornavirus VPg uridylylation complex.     

Distinct Conformations of a Putative Translocation Element in Poliovirus Polymerase

The mechanism whereby RNA is translocated by the single subunit viral RNA-dependent RNA polymerases is not yet understood. These enzymes lack homologs of the “O-helix” structures and associated fingers domain movements thought to be responsible for translocation in many DNA-templated polymerases. The structures of multiple picornavirus polymerase elongation complexes suggest that these enzymes use a different molecular mechanism where translocation is not strongly coupled to the opening of the active site following catalysis. Here we present the 2.0- to 2.6-Å-resolution crystal structures and biochemical data for 12 poliovirus polymerase mutants that together show how proper enzyme functions and translocation activity requires conformational flexibility of a loop sequence in the palm domain B-motif. Within the loop, the Ser288-Gly289-Cys290 sequence is shown to play a major role in the catalytic cycle based on RNA binding, processive elongation activity, and single nucleotide incorporation assays. The structures show that Ser288 forms a key hydrogen bond with Asp238, the backbone flexibility of Gly289 is required for translocation competency, and Cys290 modulates the overall elongation activity of the enzyme. Some conformations of the loop represent likely intermediates on the way to forming the catalytically competent closed active site, while others are consistent with a role in promoting translocation of the nascent base pair out of the active site. The loop structure and key residues surrounding it are highly conserved, suggesting that the structural dynamics we observe in poliovirus 3D^pol are a common feature of viral RNA-dependent RNA polymerases.     

Manganese-dependent polioviruses caused by mutations within the viral polymerase

Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3D(pol), we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn(2+)-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.     

An open conformation determined by a structural switch for 2A protease from coxsackievirus A16

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported “closed” state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two “switcher” residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1′, P2 and P4 are essential for substrate specificity, which was verifi ed by our substrate binding model. In addition, we compared the in vitro cleavage effi ciency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fl uorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specifi city. These new insights should facilitate the future rational design of effi cient 2A protease inhibitors.     

The RNA Template Channel of the RNA-Dependent RNA Polymerase as a Target for Development of Antiviral Therapy of Multiple Genera within a Virus Family

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2''-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3D^pol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3D^pol, (ii) had reduced activity against EMCV 3D^pol with the resistance mutations, and (iii) was most efficient in inhibiting 3D^pol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3D^pol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3D^pol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.     

Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved ‘O-helix’ of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.     

Poliovirus RNA-dependent RNA polymerase (3Dpol): kinetic, thermodynamic, and structural analysis of ribonucleotide selection

We have performed a kinetic and thermodynamic analysis of 3D(pol) derivatives containing substitutions in the ribose-binding pocket with ATP analogues containing correct and incorrect sugar configurations. We find that Asp-238, a residue in structural motif A that is conserved in all RNA-dependent RNA polymerases, is a key determinant of polymerase fidelity. Alterations in the position of the Asp-238 side chain destabilize the catalytically competent 3D(pol)-primer/template-NTP complex and reduce the efficiency of phosphoryl transfer. The reduction in phosphoryl transfer may be a reflection of increased mobility of other residues in motif A that are required for stabilizing the triphosphate moiety of the nucleotide substrate in the active conformation. We present a structural model to explain how Asp-238 functions to select nucleotides with a correct sugar configuration and a correct base. We propose that this mechanism is employed by all RNA-dependent RNA polymerases. We discuss the possibility that all nucleic acid polymerases with the canonical "palm"-based active site employ a similar mechanism to maximize fidelity.     

phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases

By multiple sequence alignments of DNA polymerases from the eukaryotic-type (family B) subgroup of protein-primed DNA polymerases we have identified five positively charged amino acids, specifically conserved, located N-terminally to the (S/T)Lx(2)h motif. Here, we have studied, by site-directed mutagenesis, the functional role of phi29 DNA polymerase residues Arg96, Lys110, Lys112, Arg113 and Lys114 in specific reactions dependent on a protein-priming event. Mutations introduced at residues Arg96, Arg113 and Lys114 and to a lower extent Lys110 and Lys112, showed a defective protein-primed initiation step. Analysis of the interaction with double-stranded DNA and terminal protein (TP) displayed by mutant derivatives R96A, K110A, K112A, R113A and K114A allows us to conclude that phi29 DNA polymerase residue Arg96 is an important DNA/TP-ligand residue, essential to form stable DNA polymerase/DNA(TP) complexes, while residues Lys110, Lys112 and Arg113 could be playing a role in establishing contacts with the TP-DNA template during the first step of DNA replication. The importance of residue Lys114 to make a functionally active DNA polymerase/TP complex is also discussed. These results, together with the high degree of conservation of those residues among protein-primed DNA polymerases, strongly suggest a functional role of those amino acids in establishing the appropriate interactions with DNA polymerase substrates, DNA and TP, to successfully accomplish the first steps of TP-DNA replication.     

Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1

Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO(3)) from 3'-phosphoadenosine 5'-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate. This structure reveals residues critical for 3'-phosphoadenosine 5'-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.     

Crystal Structure of the Full-Length Japanese Encephalitis Virus NS5 Reveals a Conserved Methyltransferase-Polymerase Interface

The flavivirus NS5 harbors a methyltransferase (MTase) in its N-terminal ≈265 residues and an RNA-dependent RNA polymerase (RdRP) within the C-terminal part. One of the major interests and challenges in NS5 is to understand the interplay between RdRP and MTase as a unique natural fusion protein in viral genome replication and cap formation. Here, we report the first crystal structure of the full-length flavivirus NS5 from Japanese encephalitis virus. The structure completes the vision for polymerase motifs F and G, and depicts defined intra-molecular interactions between RdRP and MTase. Key hydrophobic residues in the RdRP-MTase interface are highly conserved in flaviviruses, indicating the biological relevance of the observed conformation. Our work paves the way for further dissection of the inter-regulations of the essential enzymatic activities of NS5 and exploration of possible other conformations of NS5 under different circumstances.     

The crystal structure of annexin Gh1 from Gossypium hirsutum reveals an unusual S3 cluster.

The three-dimensional crystal structure of recombinant annexin Gh1 from Gossypium hirsutum (cotton fibre) has been determined and refined to the final R-factor of 0.219 at the resolution of 2.1 A. This plant annexin consists of the typical 'annexin fold' and is similar to the previously solved bell pepper annexin Anx24(Ca32), but significant differences are seen when compared to the structure of nonplant annexins. A comparison with the structure of the mammalian annexin AnxA5 indicates that canonical calcium binding is geometrically possible within the membrane loops in domains I and II of Anx(Gh1) in their present conformation. All plant annexins possess a conserved tryptophan residue in the AB loop of the first domain; this residue was found to adopt both a loop-in and a loop-out conformation in the bell pepper annexin Anx24(Ca32). In Anx(Gh1), the conserved tryptophan residue is in a surface-exposed position, half way between both conformations observed in Anx24(Ca32). The present structure reveals an unusual sulfur cluster formed by two cysteines and a methionine in domains II and III, respectively. While both cysteines adopt the reduced thiolate forms and are separated by a distance of about 5.5 A, the sulfur atom of the methionine residue is in their close vicinity and apparently interacts with both cysteine sulfur atoms. While the cysteine residues are conserved in at least five plant annexins and in several mammalian members of the annexin family of proteins, the methionine residue is conserved only in three plant proteins. Several of these annexins carrying the conserved residues have been implicated in oxidative stress response. We therefore hypothesize that the cysteine motif found in the present structure, or possibly even the entire sulfur cluster, forms the molecular basis for annexin function in oxidative stress response.