doi:10.1016/j.peptides.2006.01.018
Copyright © 2006 Elsevier Inc. All rights reserved.
NMR structure of the viral peptide linked to the genome (VPg) of poliovirus
References and further reading may be available for this article. To view references and further reading you must
purchase this article.
Catherine H. Scheina, 
, 
, Numan Oezguena, David E. Volka, Ravindranath Garimellaa, Aniko Paulb and Werner Brauna
aSealy Center for Structural Biology and Molecular Biophysics, Department of Human Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-0857, USA
bDepartment of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11790, USA
Received 10 November 2005;
revised 24 January 2006;
accepted 24 January 2006.
Available online 15 March 2006.
Abstract
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 Å RMSD (versus 6.6 Å 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.
Keywords: Viral replication; Polymerase interaction; Picornavirus; Circular dichroism; Trimethylamine N-oxide (TMAO); Solvent stabilization; Uridylylation; Post-translational modification
Fig. 1. Circular dichroism spectra of chemically synthesized VPg of poliovirus serotype 1 in two solvent conditions. In 10 mM Na phosphate buffer, pH 7 at a concentration of 0.04 mM (top line), the peptide shows little evidence of stable secondary structure. The spectrum of a 
0.2 mM solution in 1 M TMAO (bottom line) indicates the peptide is ordered. The minimum at 207 nm and the trough between 220 and 230 nm suggests a combination of secondary structure elements, with some helical content.
 |
Fig. 2. TMAO stabilizes structure based interactions between residues, as indicated by the enhanced size and number of peaks in NOESY spectra. (a) The amide proton interaction regions (N–N) of NOESY spectra in buffer without (left) and with 1 M TMAO (right) are shown. The presence of several peaks in both spectra (arrows) indicate TMAO stabilizes a peptide configuration that is already present, but at a lower concentration, in buffer not containing TMAO. (b) Fingerprint region of NOESY spectra in buffer without (left) and with (right) TMAO. Note: Interresidue cross peaks clearly visible in the TMAO spectrum (such as between backbone amides and the three prolines, Gly5, the Hβ of the reactive tyrosine, and the H
of Arg 17) are seen only at very low contour level in the buffered sample. Thus the buffer spectrum pictures were made at a lower contour level (higher sensitivity but more background peaks) than those for TMAO, to better highlight peaks common to both spectra. Background peaks can be distinguished by their low intensity, repeating patterns and their lack of correlation with any of the chemical shifts for the peptide protons. Table 2 gives a more exact quantitation of the intensity difference between peaks common to the two spectra.
Fig. 3. Comparison of the structures calculated for PV-VPg in buffer (a) and in the presence of 1 M TMAO. (b) The top 10 structures in buffer alone show much more conformational flexibility, both in the backbone and in the position of the conserved side chains. (c) Inter-residue constraints (automatically assigned) used in determining the VPg structure in TMAO.
Fig. 4. Inter-residue NOE connectivities support the helix at the C-terminus of the structure shown in Fig. 3b. (Peaks were hand assigned, from the NOE spectrum in 1 M TMAO, described in Fig. 2.)
 |
Fig. 5. Results of docking studies with the stabilized VPg structure. (A) The stabilized VPg structure docks near amino acids known to be in the interaction site (Glu382, red, Arg379 blue, Phe377 gray [31]) on the surface of the poliovirus 3Dpol. The polymerase is shown in surface representation and other nearby acidic residues (Glu369, Asp358, red) are highlighted and labeled. The VPg is in ribbon format, with Tyr3 space filling and colored gold, and the positively charged conserved sidechains (Lys 9,10, 20 and Arg17) shown in neon and colored turquoise. (B) Possible binding modes for UTP (top) and ATP on VPg. The VPg structure is shown in ribbon format with the side chains (in neon) differentially colored, and the nucleotides are in CPK format. Consistent with the net positive charge of the adenine base, interactions of ATP with VPg are primarily through its triphosphate tail. The negatively charged uridine base has many interactions with the VPg sidechains, particularly to the Lys9 and the amides of the asparagines residues, in addition to interactions between the phosphate groups and Lys20. As the top dockings varied somewhat in the exact positioning of the nucleotides, the complexes selected for this figure have a short distance between the α-phosphate of the docked nucleotide and the Tyr3-OH group. This was complex 5 for UTP (Z-dock score18.7) and complex 2 for ATP (Zdock score 38.41). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Table 1.
NMR and refinement statistics for the structure of PV-VPg in 1 M TMAO
a Pairwise RMSD was calculated among 10 refined structures.
Table 2.
The ratio of NOE cross peak intensities to that of the diagonal peaks is enhanced by the presence of 1 M TMAO
This indicates that the co-solvent increases the amount of structured peptide, and has other effects besides decreasing the exchange rate with the solvent.
Table 3.
Picornavirus VPgs have conserved sequence features
Bold capital letters: essential for poliovirus replication according to mutagenesis studies [36].
Corresponding author. Tel.: +1 409 747 6843; fax: +1 409 747 6000.
Abstract
Purchase PDF (558 K)
E-mail Article
Add to my Quick Links
Cited By in Scopus (5)
ON 
S−
PDF (328 K)
