PDBsum entry 1ra7

Transferase

PDB id: 1ra7

Contents

Protein chain

461 a.a. *

Ligands

GTP

ACY ×7

Waters ×263

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural basis for proteolysis-Dependent activation of the poliovirus RNA-Dependent RNA polymerase.

Authors

A.A.Thompson, O.B.Peersen.

Ref.

EMBO J, 2004, 23, 3462-3471. [DOI no: 10.1038/sj.emboj.7600357]

PubMed id

15306852

Abstract

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.

Figure 1.
Figure 1 Overview of poliovirus 3D^pol RdRp structure. (A) Comparison of the original partial structure (yellow) with the complete structure shown with the fingers domain in red, the palm in gray, the thumb in blue, and the active site colored magenta. The N-terminal strand (residues 12 -36) of the original structure that descended toward the active site is shown in green. The two structures were superimposed using the backbone atoms of the active site GDD motif and three residues on either side of it (i.e. residues 324 -332). (B) Superimposition of the thumb domains from the original structure (yellow) and new complete structure (blue) showing that the thumb structure is largely unchanged by the two mutations (L446D and R455D) used to break Interface I and crystallize 3D^pol in a new lattice. The side chains of Phe30 and Phe34 are shown in green for the original structure and red for the new complete structure. (C) Top view of the complete 3D^pol structure highlighting the individual fingers of the fingers domain. The index finger is shown in green, the middle finger in orange, the ring finger in yellow, and the pinky finger in pink. As in (A), the palm is shown in gray, the thumb is in blue, and the active site is colored magenta. Phe30 and Phe34 are shown as sticks, Pro119 on the pinky finger is indicated with spheres, and glycines 117 and 124 are colored in cyan. (D) Bar representation of the 3D^pol sequence colored according to the structural elements shown in (C). Sections of the sequence in the palm are in gray and the numbers correspond to the first residue in a given structural motif.

Figure 3.
Figure 3 Molecular details of the 3D^pol nucleotide-binding site illustrating how the buried N-terminus positions Asp238 for interactions with the 2' OH group of the bound NTP. (A) Superposition of three 3D^pol structures showing the selective 1.4 Å movement of Asp238 toward the active site when the N-terminus is properly positioned. The original partial wild-type structure is in pink, the 3D^pol 68/L446A/R455D structure is in salmon, and the complete structure is colored by atom type with carbons colored according to structural motifs as in Figure 1C. Most side chains have been omitted for clarity and residues 324 -332 of the active site (magenta) were used for the superimpositions. (B) Electron density map and model of the GTP molecule bound to 3D^pol with the 2' OH group making a 2.8 Å long hydrogen bond with Asp238. The GTP makes bridging interactions between the fingers and palm domains. The base is staked on Arg174 from the ring finger, the ribose interacts with Arg174 from the ring finger and Asp238 in the palm, and the triphosphate interacts with Arg163 and Lys167 from the ring finger and the backbone of the palm domain. The map is a 2.35 Å resolution 2F[o] -F[c] simulated annealing (1500 K) composite omit map contoured at 1.6 around the rGTP molecule bound after soaking crystals in 10 mM GTP. (C) Stereo view showing how the buried N-terminus of 3D^pol positions Asp238 for rNTP interactions. The N-terminus forms three hydrogen bonds with the carbonyl oxygens of residues 64, 239, and 241 (magenta bonds) that act to position Asp238 for interaction with the 2' OH of rNTPs. The structures of the G1A mutant (orange), D238A mutant (teal, only residues 238 -241 are shown), and original partial structure without a buried N-terminus (red) are superimposed using the active site.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2004, 23, 3462-3471) copyright 2004.