In situ spatial organization of Potato virus A coat protein subunits as assessed by tritium bombardment

Abstract

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing alpha-helices and beta-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two beta-strands, (ii) a C-terminal region including two alpha-helices, as well as three beta-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four alpha-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

FIG. 1

Amino acid sequence of PVA coat protein according to Puurand et al. (26) (EMBL accession no. ZZ21670). T1 to T24 are tryptic peptides, marked by double-headed arrows.

FIG. 2

Schematic drawing of the coat protein subunit secondary structure of PVA, showing the distribution of α-helices (indicated by cylinders) and β-strands (indicated by arrows).

FIG. 3

CD spectrum of PVA coat protein. The spectrum of LiCl-purified protein (90 μg/ml) in 10 mM ammonium bicarbonate buffer, pH 7.8, was measured in a 1-mm cuvette at 25°C.

FIG. 4

Distribution of the tritium labels in the coat protein (A and B) upon tritium bombardment of PVA particles. I_i/I_Σ (%), ratio of the specific radioactivity of the given amino acid (I_i) to the total specific radioactivity of the protein (I_Σ) expressed as a percentage. The number of the amino acid residue (from the N terminus) is given along the abscissa, and the predicted α-helices and β-strands are indicated below.

FIG. 5

Schematic representation of the overall fold of the PVA coat protein. α-Helices are shown as cylinders, and β-strands are shown as angled open arrows, with thick solid lines indicating interconnecting loops and the N and C termini of the polypeptide chain.