Crystal structure of coxsackievirus B3 3Dpol highlights the functional importance of residue 5 in picornavirus polymerases

Abstract

The crystal structure of the coxsackievirus B3 polymerase has been solved at 2.25-A resolution and is shown to be highly homologous to polymerases from poliovirus, rhinovirus, and foot-and-mouth disease viruses. Together, these structures highlight several conserved structural elements in picornaviral polymerases, including a proteolytic activation-dependent N-terminal structure that is essential for full activity. Interestingly, a comparison of all of the picornaviral polymerase structures shows an unusual conformation for residue 5, which is always located at a distortion in the beta-strand composed of residues 1 to 8. In our earlier structure of the poliovirus polymerase, we attributed this conformation to a crystal packing artifact, but the observation that this conformation is conserved among picornaviruses led us to examine the role of this residue in further detail. Here we use coxsackievirus polymerase to show that elongation activity correlates with the hydrophobicity of residue 5 and, surprisingly, more hydrophobic residues result in higher activity. Based on structural analysis, we propose that this residue becomes buried during the nucleotide repositioning step that occurs prior to phosphoryl transfer. We present a model in which the buried N terminus observed in all picornaviral polymerases is essential for stabilizing the structure during this conformational change.

FIG. 1.

Comparison of picornaviral polymerase structures. (A) Structures of coxsackievirus (multicolored) and poliovirus (tan) polymerases superimposed using a maximum-likelihood alignment of all Cα atoms that inherently emphasizes the palm domain, the most conserved part of the structure (18). (B) Superposition of the coxsackievirus, poliovirus (tan), and rhinovirus (light blue) polymerases showing the strong structural conservation of these enzymes and the single site of insertions and deletions in their sequences (magenta circle). The CV 3D^pol structure is colored with the convention previously used for poliovirus 3D^pol (20); palm in gray, thumb in blue, index finger in green, middle finger in orange, ring finger in yellow, and pinky finger in red. The N terminus of the CV protein is shown as a blue sphere. The view is from the left side of panel A, and the same helix is marked with an asterisk in both panels as a visual guide.

FIG. 2.

Tryptophan fluorescence spectra of wild-type coxsackievirus 3D^pol and the F5W, F5L, and F5V mutants. The F5W-wild-type difference spectrum shows that the introduced Trp5 residue has a peak at ∼350 nm that is indicative of a solvent-exposed tryptophan whose total intensity is consistent with adding an eighth Trp residue to seven already found in the protein. Spectra were collected using ∼1 μM protein and normalized to the exact protein concentration based on sample absorbance (range, 0.86 to 1.12 μM).

FIG. 3.

Structural conservation of surface-exposed residue 5 in picornaviral polymerases. The structures of poliovirus 3D^pol with a bound GTP (PDB code 1RA7), coxsackievirus B3 (3DDK), rhinovirus 1B (1XR6), and foot-and-mouth disease virus (1U09) polymerases are shown in identical views from the back of the polymerase using the same domain coloring scheme as in Fig. 1. Residue 5 is labeled in each structure, as is the location of Tyr62 in poliovirus, coxsackievirus, and rhinovirus 3D^pol and the structurally equivalent His64 in foot-and-mouth disease virus polymerase.

FIG. 4.

Interactions surrounding the polymerase N termini in 3D^pol and 3CD^pro structures. (A) Coxsackievirus 3D^pol structure showing the hydrogen bonding interactions surrounding the buried N terminus and initial β-strand. In this structure there are seven hydrogen bonds (magenta) involving residues 1 to 4 that would serve to anchor the N-terminal sequence and four hydrogen bonds (cyan) that would have to be broken in order to undergo the proposed conformational change to bury residue 5 during NTP repositioning. (B) Comparison of the poliovirus 3D^pol (multicolor) and 3CD^pro (tan) structures at the polyprotein junction showing how the polymerase domain structures are essentially identical starting at residue Ile3. The lack of the 3D^pol N terminus in the uncleaved precursor protein means that the first two residues of the polymerase become part of the flexible interdomain linker between the 3C^pro and 3D^pol domains (cyan with a blue sphere marking the position of the polymerase domain Gly1 amino group). As a result, only one of the seven N-terminal hydrogen bonds found in 3D^pol is formed in the 3CD^pro structure (marked with an asterisk in panel A). The superpositioning of the 3D^pol and 3CD^pro structures was based on the entire polymerase domain and is dominated by the palm structure (as shown in Fig. 1), resulting in a slight rotational offset between 3D^pol and 3CD^pro in this region of the structures.