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• W2000594526 abstract "Article2 February 2006free access The structure of a protein primer–polymerase complex in the initiation of genome replication Cristina Ferrer-Orta Cristina Ferrer-Orta Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Armando Arias Armando Arias Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Rubén Agudo Rubén Agudo Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Rosa Pérez-Luque Rosa Pérez-Luque Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Cristina Escarmís Cristina Escarmís Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Esteban Domingo Esteban Domingo Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Nuria Verdaguer Corresponding Author Nuria Verdaguer Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Cristina Ferrer-Orta Cristina Ferrer-Orta Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Armando Arias Armando Arias Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Rubén Agudo Rubén Agudo Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Rosa Pérez-Luque Rosa Pérez-Luque Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Cristina Escarmís Cristina Escarmís Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Esteban Domingo Esteban Domingo Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain Search for more papers by this author Nuria Verdaguer Corresponding Author Nuria Verdaguer Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain Search for more papers by this author Author Information Cristina Ferrer-Orta1,‡, Armando Arias2,‡, Rubén Agudo2, Rosa Pérez-Luque1, Cristina Escarmís2, Esteban Domingo2 and Nuria Verdaguer 1 1Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Barcelona, Spain 2Centro de Biologia Molecular ‘Severo Ochoa’ (CSIC-UAM), Cantoblanco, Madrid, Spain ‡These authors contributed equally to this work *Corresponding author. Institut de Biologia Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain. Tel.: +34 93 403 49 52; Fax: +34 93 403 49 79; E-mail: [email protected] The EMBO Journal (2006)25:880-888https://doi.org/10.1038/sj.emboj.7600971 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix α8 of the fingers domain and helix α13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction. Introduction A number of DNA viruses, RNA viruses and linear plasmids use small proteins as primers for DNA or RNA synthesis (Salas, 1991). These proteins are covalently attached to the 5′ end of the DNA or RNA genomes. During initiation of replication, they provide the hydroxyl group required by the polymerase to start polynucleotide synthesis. In the process of replication, the first step is the linkage of a nucleotide to the hydroxyl group of an amino acid in the primer protein. This process is catalyzed by the DNA or RNA polymerase and the same enzymes are also responsible for the elongation of the DNA or RNA strands. Thus, these polymerases are structurally adapted for using either a protein or a nucleic acid as a primer. Picornaviruses use a protein of 20–24 amino acids, termed VPg, to initiate viral RNA synthesis (Paul, 2002). The different picornaviruses show partial amino-acid sequence identities in their VPgs that range from 17 to 26% (http://www.iah.bbsrc.ac.uk/virus/picornaviridae/SequenceDatabase/alignments/pico2c3d_pep.txt). Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family, possesses a linear plus strand RNA genome about 8500 nucleotides long. It contains a long 5′-untranslated region (UTR), a single open reading frame and a short 3′-UTR with a poly(A) tail. The 5′-terminal uridine of the RNA is covalently linked to the hydroxyl group of a tyrosine in the terminal protein VPg. The genome of FMDV encodes three different VPgs (VPg1, VPg2 and VPg3) in tandem (Forss and Shaller, 1982), and all three are active as primers for RNA replication. Deletion of any individual copy of VPg has a deleterious effect on RNA replication (Falk et al, 1992). The replication of this RNA requires all of the nonstructural proteins of the virus and also several cis-replicating elements (Nayak et al, 2005). The viral proteins most directly involved in RNA synthesis are the template- and primer-dependent RNA polymerase (3D), the terminal protein VPg and 3CD (unprocessed 3C–3D), which acts as a proteinase and an RNA binding protein (Paul, 2002; Nayak et al, 2005). We have previously reported the structure and interactions of the FMDV 3D polymerase with a template–primer RNA substrate, providing insights of the RNA elongation process and the interactions of the FMDV 3D with the exiting duplex product (Ferrer-Orta et al, 2004). In addition, modeling studies based on alignments of the FMDV 3D/RNA complex with the complexes HIV-RT/DNA and phi6-RdRP/DNA (Huang et al, 1998; Butcher et al, 2001) allowed us to identify several key residues in the FMDV-3D polymerase, which may be involved in nucleotide (NTP) binding and catalysis. In our model, the conserved residues Asp338 and Asp339 of motif C, together with Asp245 in motif A, are in the adequate position to bind to the divalent cations, helping to orient the 5′ phosphate group of the incoming nucleotide for nucleophilic attack by the 3′ end of the primer. The basic residues Arg168, Lys172 and Arg179 of motif F are also in an ideal position to interact with the negatively charged phosphates of the nucleotide substrate (Ferrer-Orta et al, 2004). The mechanism of the critical event of initiation of RNA synthesis during picornavirus replication is largely unknown owing to the absence of structural information. Here, we report the 3D structure of two complexes between the FMDV polymerase (3D) and its protein–primer VPg1 in its nonuridylylated and uridylylated forms. The structures reveal critical VPg and 3D amino acids and the interactions involved in the positioning and addition of the first nucleotide (UMP) to the VPg molecule. In addition, the complex with the uridylylated VPg1 form clarifies the role of the metal ions in the priming reaction and provides new insights into the mechanism of initiation of RNA genome replication. Amino-acid replacements at several positions of 3D and VPg resulted in the modification of uridylylation activity, in agreement with the structure of the complexes. Results Structure of the VPg primer protein bound to the 3D polymerase Two different crystal forms of the complex between the FMDV 3D polymerase, the primer protein VPg, UTP and metal ions, in the absence and in the presence of an RNA oligonucleotide of 10 adenine bases (A10) were obtained and analyzed by X-ray crystallography. The structures of the complexes were obtained at 3.0 and 2.9 Å, respectively (Table I). In the two crystal forms, the quality of the electron density maps, after several rounds of manual modeling and refinement, allowed the accurate positioning of 15 of the 23 amino acids of the VPg protein (Figure 1). Weak and discontinuous density was also visible to accommodate one or two additional VPg residues poorly ordered, exiting from the polymerase. The remaining six carboxy-terminal residues of VPg appeared completely disordered. Figure 1.Structure of the primer protein VPg in a complex with 3D. (A) Stereo view of a sigma A weighted ∣Fo∣−∣Fc∣ electron density map at 2.9 Å resolution and contoured at 3.0σ around the VPg–UMP molecule (The VPg–UMP and ions were omitted from the phasing model). The 15 amino acids of VPg, the UMP covalently linked to the protein and the metal ions are placed inside the density in ball and stick representation colored in atom type code. Names for all residues are explicitly labeled in one letter code. (B) Details of the interactions seen in the active site of the 3D polymerase during the uridylylation reaction. The residues Pro2, Tyr3 and Ala4 of VPg are shown in sticks in red and the UMP, covalently linked to the hydroxyl group of Tyr3, in light green. The divalent cations Mn2+ and Mg2+ are shown as magenta and orange spheres, respectively, and the anomalous difference Fourier map is shown as a chicken wire in blue. The 3D amino acids involved in direct hydrogen bonds with ions and the uridylylated tyrosine are shown in ball and sticks in atom type code, and the hydrogen bonds appear as dashed lines. All residues are explicitly labeled. The predicted position of the oligo(A) template strand (dark green) was determined using the 3D–RNA template–primer complex (PDB entry 1WNE) as a guide. Download figure Download PowerPoint Table 1. Data collection and refinement statistics 3D+VPg 3D+VPg+UMP 3D+VPg+UMPa Data collection details Space group P3221 P3221 P3221 Unit cell (Å) a=b=95.09, c=100.63 a=b=94.38, c=99.71 a=b=94.530, c=100.403 Wavelength (Å) 0.93 0.98 1.89 Resolution rangeb (Å) 30–3.0 (3.1–3.0) 30–2.9 (3.0–2.9) 30–3.1 (3.2–3.1) 〈I/σ〉 6.8 (2.3) 7.5 (2.8) 12.2 (7.5) Rmergec 9.5 (40.5) 9.5 (45.5) 6.1 (37.2) Completeness (%) 100 (100) 99.9 (99.9) 100 (99.9) Refinement statistics Unique data 10 830 11 795 Rworkd (%) 27.7 26.9 Rfreed (%) 29.4 28.5 No residues 3D pol 474 474 VPg 15 15 Metal ions — Mn2+ and Mg2+ Average temperature factors (Å2) All atoms 76.9 65.8 3D pol 76.8 65.5 VPg proteine 81.9 66.4 Metal ionse — 66.7 aData collected at the Mn K edge and used only to calculate the anomalous scattering difference Fourier map. bData within parenthesis are for the highest resolution shell. cRmerge=∑j∑h (∣Ij,h−〈Ih〉∣)/∑j∑h (〈Ih〉), where h are unique reflections indices, Ij,h are intensities of symmetry-related reflections and 〈Ih〉 is the mean intensity. dRwork and Rfree are defined by R=∑hkl∣∣Fobs∣−∣Fcalc∣∣/∑hkl∣Fobs∣, where h, k and l are the indices of the reflections (used in refinement for Rwork; 5%, not used in refinement, for Rfree), and Fobs and Fcalc are the structure factors, deduced from measured intensities and calculated from the model, respectively. eThe VPg protein and metal ions were refined with occupancy of 70%. The 15 VPg amino acids determined in both structures showed almost the same conformation with little secondary structure. The N-terminal portion of the protein is located close to the NTP entry cavity and projects the side chain of residue Tyr3 into the active site. Then, the peptide chain snakes through the large RNA binding cleft towards the thumb domain of the 3D protein, following a similar trajectory to that taken by the RNA primer and duplex product in the 3D–RNA complex (Ferrer-Orta et al, 2004; Figure 2). Figure 2.Structure of VPg bound to the FMDV 3D polymerase. (A) Molecular surface of the 3D polymerase shown in two different views: the conventional orientation, as if looking into a right hand (left) and a side view (right). The polymerase domains: fingers, palm and thumb are colored in light-blue, dark-blue and purple, respectively. The VPg protein that binds across the RNA binding cleft is represented as a red ribbon with side chains shown in stick representation in red, the UMP molecule covalently linked to the hydroxyl group of the VPg residue Tyr3 is shown in green, and the metal ions as orange spheres. In the right panel, the fingers residues at the top of the NTP tunnel and most of the thumb domain are removed to allow the visualization of VPg. (B) Top-down views of the polymerase molecule showing the trajectory of the VPg protein (left) compared to the trajectory of RNA template–primer (right) in the structure of the complex FMDV 3D–RNA template–primer (PDB entry 1WNE; Ferrer-Orta et al, 2004). Comparisons were carried out by the structural superimposition of both polymerase complexes that gave a root mean square deviation of 0.33 Å for the superimposition of all Cα atoms. The RNA is shown as a ribbon representation in green (template chain) and yellow (primer chain). Roughly, the N-terminal moiety of the bound VPg protein occupies part of the NTP entry channel and the position of the primer, whereas the carboxy-terminus of the protein superimposes with the trajectory of the RNA duplex product. In both panels, the N-terminal residues (from 34 to 48) and residues at the top of the NTP tunnel (from 163 to 180) of 3D are omitted to better show the VPg protein. Download figure Download PowerPoint In the crystals obtained when the oligonucleotide A10 was absent, no density was observed to accommodate the UTP substrate. However, in the complex obtained in the presence of A10 and MgCl2, the difference in electron density maps (2∣Fo∣−∣Fc∣ and ∣Fo∣−∣Fc∣) showed the product of the VPg uridylylation (VPg-UMP) and revealed the presence of two metal ions in the polymerase catalytic site (Figures 1A and B). To distinguish whether Mg2+ or Mn2+ was at the active site was not obvious because both cations were present during the complex formation and the crystallization processes. We have recently reported the ability of FMDV 3D polymerase to catalyze the uridylylation of VPg using poly(A) as a template in the presence of Mn2+ ions (Arias et al, 2005). In contrast, the VPg uridylylation in the presence of Mg2+ was undetectable (A Arias and R Agudo, unpublished results). In order to provide an experimental evidence of the presence of Mn2+ in the active site of our complex, we collected a new data set of the 3D–VPg–UTP–metal complex cocrystals using the wavelength of the radiation tuned to the Mn2+ K edge, 1.89 Å (see Materials and methods section and Table I). The anomalous scattering difference Fourier map showed one peak (4σ level), indicating the presence of a single Mn2+ ion bound to the active site of the polymerase. The second strong peak seen in conventional difference Fourier maps was then interpreted by the presence of one additional Mg2+ cation (Figure 1). No ordered density was visible in the template entry channel to position the oligonucleotide that was expected to act as a template in the uridylylation. Therefore, it seems that uridylylation reaction took place before crystal formation. The pyrophosphate product was also absent. The position of the oligo(A) template was further modeled using the FMDV 3D–RNA template/primer complex (Ferrer-Orta et al, 2004) as a guide (Figure 1B). The resulting model shows how the first five nucleotides of the oligo(A) template traverse the template entry channel pointing the base, A5 towards the catalytic site in a correct position, to be paired with the UMP molecule covalently attached to VPg (Figure 1B). VPg–3D polymerase interactions The structures determined show how VPg accesses the polymerase catalytic site from the front part of the molecule through the RNA binding cleft (Figure 2). No important conformation changes of the 3D polymerase conformation were induced by the VPg binding; only local rearrangements in the side chain of residues Arg179 and Asp338, which participate directly in the uridylylation reaction, were seen (see below). The largest network of VPg–3D interactions was observed between residues Glu166, Ile167, Arg168, Lys172 and Arg179, in motif F of the fingers domain, which, together with residues Thr407, Ala410 and Ile411 of helix α13 of the thumb domain, contacted the N-terminal moiety of VPg, stabilizing the conformation of Tyr3 in the active site cavity (Figure 3). Two main chain–side chain hydrogen bonds were observed between the backbone oxygen atoms of Pro2 and Tyr3 of VPg, and the amino groups of the side chains of Lys172 and Arg168 of the polymerase. The guanidinium group of Arg179, which forms a double salt bridge with Glu166 within the same motif F of 3D is also hydrogen bonded with the hydroxyl group of Tyr3 in the nonuridylylated VPg. In the complex with the uridylylated form of VPg, the side chain of Arg179 changes slightly its conformation, maintaining the interaction with Glu166 and forming an additional salt bridge with the phosphate oxygen O2 of UMP (Figures 1B and 3). The main chain oxygen atoms of Pro6 and Leu7 of VPg were hydrogen bonded to the side chain of the 3D residue Lys387, located in motif E of the palm domain. The side chain of Leu7 is also located in a hydrophobic cavity formed by the side chains of residues Glu166 and Ile167 of motif F and the side chains of Thr407, Ala410 and Ile411 of helix α13 (Figure 3). Additional contacts were observed between Arg388 of motif E and residues Glu8, Arg9 and Gln10 of VPg. The side chain of Tyr336, within the catalytic motif C, also participates in interactions with VPg forming a hydrogen bond with the guanidinium group of Arg11 of the peptide primer. Finally, the 3D residues Gly216, Cys217 and Pro219, located at the beginning of the helix α8 in the fingers domain, also establish hydrophobic contacts with VPg residue Arg11 at the exit of the polymerase cavity (Figure 3). Figure 3.VPg–3D polymerase interactions. (A) Structure of the VPg primer protein (red) with the contacting residues of the 3D polymerase shown in different colors. Four different regions of the polymerase molecule contact VPg residues E166, I167, R168, K172 and R179, belonging to motif F of fingers (orange), together with residues T407, A410 and I411 of the thumb domain (light blue), interact with the N-terminal moiety of VPg, stabilizing the conformation of Y3 in the active site cavity. In addition, residues E166, I167 of motif F (orange), K387 and R388 of motif E (dark blue) and T407, A410 and I411 of helix α13 (light blue) interact with the central part of the VPg protein. Finally, the 3D residues G216, C217 and P219, located at the beginning of helix α8 (light blue) in the fingers domain, together with the side chain of Y336 within the C motif (yellow) of the palm domain, establish hydrophobic contacts with R11 at the exit of the polymerase cavity. (B) Structure of the uridylylated VPg protein (shown in red and the linked UMP in green) with the contacting residues of the 3D polymerase shown in blue. In addition to the interactions described in (A), amino acids D245 of motif A (pink) and D338 of motif C (yellow) are placed in the correct orientation for the catalysis of the phosphodiester linkage in the active site of the 3D protein. Download figure Download PowerPoint Insights into the mechanism of VPg uridylylation The comparison of the 3D–VPg and the 3D–VPg–UMP structures reveal how the side chain of Tyr3 is oriented towards the active site close to the catalytic aspartic acids 245 of motif A and 338 of motif C. In the 3D–VPg–UMP complex, the hydroxyl group of Tyr3 side chain was found covalently attached to a UMP molecule by a phosphodiester linkage (Figure 1B). Two metal ions, Mn2+ and Mg2+, participate in the uridylylation reaction that appears to follow a similar mechanism to that described for the nucleotidyl transfer reaction in other polymerases (Steitz, 1998). Mn2+ bridges the carboxylate group of Asp338 and the O− of tyrosine side chain, now covalently bonded to phosphate α of UMP. The side chain of Asp338 changes its rotamer conformation to optimize the described interactions. Mg2+ coordinates the carboxylic group of Asp245, the O1 oxygen of phosphate α and the hydroxyl group of Ser298 side chain. Tyr336 of motif C, which is hydrogen bonded to the VPg residue Arg11, also participates in hydrophobic contacts with the UMP molecule covalently attached to VPg (Figure 3). Finally, the positively charged residues of motif F (Arg168, Lys172 and Arg179) also participate in the uridylylation process (Figure 1B). These basic amino acids of motif F, together with Tyr336 of motif C, seem to play a role stabilizing Tyr3 and UMP in a proper conformation for the catalytic reaction. Biochemical analysis of structure-based mutants of polymerase and VPg molecules To investigate the effect of amino-acid replacements in 3D and VPg in the uridylylation reaction, five single replacements (Glu166-Ala, Glu166-Arg, Arg168-Ala, Arg179-Ala, Asp338-Ala) and two double replacements (Lys387-Ala/Arg388-Ala, Thr407-Ala/Ile411-Ala) were introduced in 3D by site-directed mutagenesis of plasmid pET-28a3Dpol (Materials and methods). The uridylylation activity on VPg1 was tested and compared with that of the wild-type 3D. The results (Figure 4A) show at least a 10-fold reduction for all mutants examined, except for 3D mutant Arg168-Ala that showed about 60% of the activity relative to wild-type 3D, and the double mutation Thr407-Ala/Ile411-Ala, which did not produce any detectable effect on the uridylylation activity. Figure 4.Effect of amino acids replacements in 3D and VPg on the VPg-uridylylation activity. (A) VPg1 uridylylation activity by substituted FMDV 3Ds, including one or two amino-acid replacements, relative to the wild-type enzyme. Values are the average of at least three independent experiments. The mono- and di-uridylylated band of a representative electrophoretic separation is shown. No significant differences were seen when the reaction products were treated with RNAse A before electrophoresis. Preparation of mutant 3Ds and assay procedures are described in Materials and methods. (B) Uridylylation activity of wild-type FMDV 3D on VPg1, VPg2, VPg3 and VPg1 with single amino-acid substitution. Results and procedures are as in (A). Download figure Download PowerPoint VPg replacements Tyr3-Phe, Glu8-Ala, Arg9-Ala, Arg9-Glu and, to a lesser extent, Pro6-Ala resulted in significant decreases in uridylylation activity, as compared with wild-type VPg1, VPg2 or VPg3 (Figure 4B). Thus, modification of 3D and VPg residues, predicted to be involved in critical interactions according to the 3D structure of the uridylylation complex, have remarkable effects in the uridylylation reaction. Discussion Implications for the mechanism of initiation of RNA synthesis The 3D structures of RdRPs, unbound to ligands, are available for four other members of the Picornaviridae family (PV, HRV1B, HRV14 and HRV16) (Love et al, 2004; Thompson and Peersen, 2004; Appleby et al, 2005). The structure-based alignment of this five picornaviral polymerases shows that 12 out of the 16 residues of 3D polymerase involved in VPg binding are strictly conserved (Figure 5A). Sequence alignments of the VPg proteins from these picornaviruses also revealed a partial conservation of the 3D-interacting residues (Figure 5B). Mapping of the 12 VPg-interacting residues onto the surface of the FMDV polymerase is shown in Figure 6A. Structural comparisons between FMDV–3D–VPg complex with the other four picornaviral RdRPs showed that the size and the shape of the central cavity, where VPg binds, is almost identical in all five polymerases (Figure 6B), and FMDV VPg protein can be modeled in the central cavity of all picornaviral polymerases, without important steric hindrances. The modeled VPg in the central cavity of PV and rhinovirus RdRPs retained almost the same interactions described in the FMDV–3D–VPg complex. In particular, those relevant to the uridylylation reaction would be strictly conserved (Figures 5 and 6B). Figure 5.(A) Structure-based alignment of the picornavirus polymerase residues that make contact with VPg. Aligned are domains for the different picornaviral polymerases whose structure is known. The strictly conserved residues are in red blocks and similar residues in red characters and blue boxes. The FMDV 3D residues interacting with the VPg primer protein are marked by green inverted triangles (B) Sequence alignment of the VPg protein of the different picornaviruses. The FMDV VPg1 residues interacting with 3D are marked by green squares. Residues of PV VPg previously shown to interact with PV 3D polymerase by yeast two-hybrid analysis (Xiang et al, 1998) are highlighted in yellow blocks. Download figure Download PowerPoint Figure 6.Comparison of FMDV polymerase bound to VPg with other picornaviral polymerases. (A) Mapping of the conserved FMDV 3D residues that contact the VPg primer protein onto the polymerases structure (blue). The VPg-interacting residues are colored according to the different polymerase motifs: D245 of motif A is in magenta; E166, R168, K172 and R179 belonging to motif F are in orange; residues G216, C217 and P219 located in or near helix α8 in dark gray; amino acids Y336 and D338 of motif C are in yellow; and residues K387 and R388 of motif E in dark blue. The VPg protein occupying the central cleft of the polymerase is shown in red. (B) Stereoview of the structural superimposition of the coordinates of PV (green) and HRV16 (yellow) RdRPs onto the FMDV 3D–VPg complex (blue), showing the positioning of the VPg primer protein (red) in the central cavity of the polymerases. The Cα root mean square deviation is 1.24 Å for the superimposition of 362 atoms between PV and FMDV RdRPs, and 1.29, 1.25 and 1.29 Å for the superimpositions of 254, 370 and 353 Cα atoms, between FMDV and HRV1B, HRV14 and HRV16 RdRPs, respectively. The conserved VPg-interacting residues are shown as in (A). (C) Ribbon diagram of the structure of PV 3D polymerase (green) with the VPg of FMDV modeled inside (red). Two different views of the PV polymerase are shown: the conventional orientation (left) and a side view (right; the finger residues at the top of the NTP tunnel and most of the thumb domain are removed to allow the visualization of VPg). The residues that affected VPg binding (yellow) and uridylylation (blue), as determined by mutational analysis (Lyle et al, 2002), are shown as stick representation and are explicitly labeled. In the model, Tyr326 has the correct orientation to interact directly with the UMP substrate, and Lys359 is properly oriented to interact directly with the N-terminal moiety of the VPg peptide. Download figure Download PowerPoint The FMDV mutants at the conserved polymerase residues that strongly interact with VPg—Glu166 and Arg179 of motif F, Asp338 of motif C and Lys387/Arg388 of motif E—show a drastic defect in VPg uridylylation (Figure 4). Substitution of Arg168 resulted in ∼40% reduction in uridylylation. This residue contacts VPg through a hydrogen bond with the backbone oxygen atom of Tyr3. The lack of the Arg168 side chain can be, at least in part, compensated by the presence in the vicinity of other positively charged side chains, like that of Lys172 (Figure 3). The double mutation Thr407/Ile411 to alanine shows no defect in VPg uridylylation (Figure 4). These residues form part of a hydrophobic cavity that accommodates Leu7 of VPg (Figure 3). It seems that alanine residues do not disturb this hydrophobic cavity. Both residues are not conserved among picornaviruses. Mutational analysis in PV has identified two groups of residues at the surface of the polymerase whose substitutions affected either the rate of VPg uridylylation or the 3AB binding affinity and the ability of membrane-bound 3AB recruitment. Uridylylation was also partially affected by this second group of mutants (Lyle et al, 2002; Boerner et al, 2005; Figure 6C). Substitution of the first group of residues (Tyr326, Asp358 and Lys359) corresponding to FMDV (Tyr336, Asp368 and Lys369) results in a drastic loss of uridylylation activity (Lyle et al, 2002). These residues are located at or near the VPg binding site (Figure 6C). The second group of residues (Phe377, Arg379, Glu382 and Val391) that would correspond to His389, His391, Tyr394 and Val402 in FMDV are located on the back side of the polymerase and lie far from VPg to allow direct contacts with this primer protein (Figure 6C). Substitution of most amino acids of this second group resulted only in a moderate decrease of VPg uridylylation activity, perhaps because this major effect is on binding of the 3AB precursor rather than of VPg (3B). Additional studies are needed to clarify this point" @default.
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• W2000594526 date "2006-02-02" @default.
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• W2000594526 title "The structure of a protein primer–polymerase complex in the initiation of genome replication" @default.
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• W2000594526 doi "https://doi.org/10.1038/sj.emboj.7600971" @default.
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