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PDBsum entry 2f8e
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References listed in PDB file
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Key reference
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Title
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The structure of a protein primer-Polymerase complex in the initiation of genome replication.
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Authors
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C.Ferrer-Orta,
A.Arias,
R.Agudo,
R.Pérez-Luque,
C.Escarmís,
E.Domingo,
N.Verdaguer.
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Ref.
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EMBO J, 2006,
25,
880-888.
[DOI no: ]
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PubMed id
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Abstract
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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 alpha8 of the fingers domain and helix alpha13 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.
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Figure 1.
Figure 1 Structure of the primer protein VPg in a complex with
3D. (A) Stereo view of a sigma A weighted |F[o]|-|F[c]| electron
density map at 2.9 Å resolution and contoured at 3.0  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.
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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  13
(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 8
(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.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
EMBO J
(2006,
25,
880-888)
copyright 2006.
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Secondary reference #1
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Title
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Structure of foot-And-Mouth disease virus RNA-Dependent RNA polymerase and its complex with a template-Primer RNA.
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Authors
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C.Ferrer-Orta,
A.Arias,
R.Perez-Luque,
C.Escarmís,
E.Domingo,
N.Verdaguer.
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Ref.
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J Biol Chem, 2004,
279,
47212-47221.
[DOI no: ]
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PubMed id
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Figure 3.
FIG. 3. Electron density maps around the FMDV 3D active
site. A, stereoview of the final  [A]-weighted 2F[o] -
F[c] Fourier map, contoured at 1.5 , in the isolated 3D
structure with the model placed inside (ball and sticks colored
in atom type code). B, [A]-weighted 2F[o] -
F[c] map, shown at 1.0 , in the FMDV 3D-RNA
complex structure. A portion of the RNA oligonucleotide is shown
in the picture in a stick representation in light green, the
template strand, and dark green, the primer strand. Only two
nucleotides of the template and three of the primer are shown
for clarity. The Mg2+ ion, located close to acidic residues
Asp238, Asp240, and Asp339, is shown as an orange ball. Water
molecules are shown as red balls and labeled as w.
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Figure 6.
FIG. 6. Protein-protein interactions in FMDV 3D polymerase.
A, ribbon representation of the largest interface of
interactions in the P4[1]2[1]2 crystal lattice, also conserved
in P3[2]21 crystals; B, close up of the interacting surfaces.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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