Proteome of the large Pseudomonas myovirus 201 phi 2-1: delineation of proteolytically processed virion proteins

Abstract

Pseudomonas chlororaphis phage 201 phi 2-1 produces a large structurally complex virion, including the products of 89 phage genes. Many of these proteins are modified by proteolysis during virion maturation. To delineate the proteolytic maturation process, 46 slices from an SDS-polyacrylamide gel were subjected to tryptic digestion and then HPLC-electrospray ionization-tandem mass spectrometry analysis. The scale of the experiment allowed high sequence coverage and detection of mass spectra assigned to peptides with one end produced by trypsin and the other end derived from a maturation cleavage (semitryptic peptides). Nineteen cleavage sites were detected in this way. From these sites, a cleavage motif was defined and used to predict the remaining cleavages required to explain the gel mobility of the processed polypeptide species. Profiling the gel with spectrum counts for specific polypeptide regions was found to be helpful in deducing the patterns of proteolysis. A total of 29 cleaved polypeptides derived from 19 gene products were thus detected in the mature 201 phi 2-1 virion. When combined with bioinformatics analyses, these results revealed the presence of head protein-encoding gene modules. Most of the propeptides that were removed from the virion after processing were acidic, whereas the mature domain remaining in the virion was nearly charge-neutral. For four of these processed virion proteins, the portions remaining in the mature virion were mutually homologous. Spectrum counts were found to overestimate the relative quantity of minor polypeptide species in the virion. The resulting sensitivity for minor species made it possible to observe a small amount of general proteolysis that also affected the virions.

Fig. 1.

SDS-PAGE of 201φ2-1 virion proteins. The boundaries of slices that were digested and analyzed by MS are shown immediately to the right of the gel. Each virion protein is listed next to the slice containing its maximum SC. For virion proteins listed in red, the polypeptide has been altered by proteolytic cleavage by the prohead protease. In cases where more than one polypeptide segment remained in the virion, the segments have been annotated with N, M, or C to indicate N-terminal, middle (if present), and C-terminal segments, respectively.

Fig. 2.

Analysis of gp455 proteolytic processing. A, SC gel profile. Detected peptides have been subdivided into N-terminal, middle, and C-terminal regions covering the residue ranges indicated. No peptides were detected in other slices or outside of the indicated ranges. B, peptide coverage. Semitryptic peptides are indicated in red. Above the plot, the bit score of prospective cleavage sites is indicated by the height of a vertical arrow. A horizontal line is drawn, dividing cleavage motifs that were considered high scoring from those considered low scoring as described in the text. The two cleavage sites indicated as “duplicated” arose through a domain duplication. Horizontal arrows indicate the inferred gp455N, -M, and -C polypeptide segments. C, correspondence of the φKZ homolog gp303. Black boxes indicate regions of similarity as detected by Blast. Vertical arrows indicate bit scores of prospective cleavage motifs. The thin line to the left is an extended N-terminal region that is not present in 201φ2-1 gp455. The connector between the two boxes indicates that this region is deleted relative to 201φ2-1 gp455.

Fig. 3.

Sequence logo representing 19 cleavage sites confirmed by detection of semitryptic peptide.

Fig. 4.

Heterogeneous processing of major capsid protein. The spectrum count of tryptic peptides assigned within slice 10 is given above the gp200 sequence. The count of 45 listed for VVTVGYNLAAS includes 20 spectra for peptides having additional residues to the left or right due to missed trypsin cleavages. Residue numbers refer to gp200. High scoring cleavage motifs are in red. Residues after which trypsin should cleave in gp200 are in blue. Cleavage at the indicated position for φKZ gp120 and EL gp78 was established by Edman degradation (11, 12).

Fig. 5.

Processing of gp200 (major capsid protein). A, SC gel profile. The protein sequence has been subdivided into the following regions: N (before the stabilization domain), M (stabilization domain including linker regions), and C (after stabilization domain). B, sequence coverage for peptides assigned at ≥95% confidence. Semitryptic peptides are shown in red and pink. The pink peptides migrated in slice 10, which would indicate a derivation from an intact gp200 molecule rather than from a cleaved species. The slice numbers of species produced by the respective cleavages are indicated below the sequence coverage plot. S17 and S44 are slices where the indicated semitryptic peptides are found. For each cleavage, the slice number of the remainder of gp200 that can be inferred to have been produced is indicated next to a dashed line. The inferred partner to the S17 species is expected to be in slice 30 if intact, but instead its components are scattered below slice 37. S10 is the slice number of gp200 cleaved only by the prohead protease. C, position of gp200 domains. The core domains are those in common with T4 and HK97 major capsid proteins, whereas the SD domain is in common with T4 but not HK97 major capsid proteins (26). Endo refers to an endothiapepsin domain peculiar to φKZ-related phages (9). D, location of non-prohead protease cleavages within the gp200 sequence.

Fig. 6.

Regions of 201φ2-1 genome encoding processed virion proteins. Genomic segments from the full GBrowse display of 201φ2-1 were juxtaposed, and protein features were mapped onto the corresponding genes. Green arrows, proteins detected by mass spectrometry; dark green arrows, virion proteins that were determined to be abundant (13); diagonally striped green arrows, virion-associated proteins newly detected in this study; orange outlined arrows, proteins for which the φKZ homologs were found to be present in a tailless mutant (15); gray arrows, non-virion proteins; purple horizontal bars, a region of sequence homology found in several 201φ2-1 proteins; red horizontal bars, a region with greater than one net negative charge per 10 residues; pink horizontal bars, a region with greater than one net negative charge per 20 residues. Peptide coverage is indicated as follows in the boxed regions below each gene glyph: green, tryptic peptide coverage; red, semitryptic peptide. Triangles above the peptide coverage boxes indicate prohead protease cleavage sites: dark blue, established by a semitryptic peptide; cyan, predicted by motif searching. Thin black arrows below the peptide coverage boxes correspond to the position of a mature polypeptide and corresponding slice number. Two alternative interpretations are shown for gp155N. RNAP, RNA polymerase.