Mutational Analysis of the Pseudomonas aeruginosa Myovirus ϕKZ Morphogenetic Protease gp175

Abstract

Pseudomonas aeruginosa myovirus KZ has a 270-kb genome within a T‫72؍‬ icosahedral capsid that contains a large, unusual, and structurally well-defined protein cylindrical inner body (IB) spanning its interior. Proteolysis forms a pivotal stage in KZ head and IB morphogenesis, with the protease gp175 cleaving at least 19 of 49 different head proteins, including the major capsid protein and five major structural IB proteins. Here we show that the purified mature form of gp175 is active and cleaves purified IB structural proteins gp93 and gp89. Expression vector synthesis and purification of the zymogen/precursor yielded an active, mature-length protease, showing independent C-terminal gp175 self-cleavage autoactivation. Mutation of either the predicted catalytic serine or histidine inactivated mature gp175, supporting its classification as a serine protease and representing the first such direct biochemical demonstration with purified protease and substrate proteins for any phage protease. These mutations also blocked self-cleavage of the precursor while allowing intermolecular gp175 processing. To confirm the cleavage specificity of gp175, we mutated three cleavage sites in gp93, which blocked proteolysis at these sites. The N-terminal propeptide of gp93 was shown to undergo more extensive proteolysis than previously identified. We found that proteolysis in gp93 progressed from the N to C terminus, while blocking cleavage sites slowed but did not eliminate downstream proteolysis. These findings were shown by informatics to be relevant to the head morphogenesis of numbers of other related IB-containing giant phages as well as to T4 and herpesviruses, which have homologous proteases.T he giant myovirus KZ, which is infective for Pseudomonas aeruginosa, is a member of the KZ-related phages that currently incorporate two genera, the KZ-like phages (KZ, PA3, and 2012-1) and the more distantly related EL-like phages (EL and OBP) (1-3). These phages are distributed widely geographically and are of interest because those infective for pathogens have been used for phage therapy (4) and/or have potential for further biocontrol applications (5-8). These phages are also a curiosity, as they are remarkably complex, both at the genome and structural levels, with large virions comprised of a Tϭ27 capsid or head (9) and contractile tail that ends in an elaborate baseplate (10, 11). The complexity of the virion is reflected in the numbers of different proteins, Ͼ60 different proteins, with nearly 50 different proteins present in the head alone, possibly the highest number for any known group of viruses (12)(13)(14). A remarkable feature of the KZ-related phages is that within the head there is a large, protein cylinder called an inner body (IB) around which the DNA is tightly spooled (15). The structure of the KZ IB was recently solved, revealing it to be approximately 24 by 105 nm and tilted at an angle of approximately 22°relative to the portal axis (16). Its structure is also regular, with an apparent 6-fold symmetry in some ...

Common Evolutionary Origin of Procapsid Proteases, Phage Tail Tubes, and Tubes of Bacterial Type VI Secretion Systems

“…The catalytic triad of T4 gp21 is formed by Ser 140 , His 85 and Asp 168 , consistent with earlier sequence analyses (Liu and Mushegian, 2004; Thomas and Black, 2013). Residues 140 and 85, which had been mutated to alanines (Figure 2), are located in β–strand 6, and in the loop connecting α-helix 1 to β–strand 4, respectively, whereas Asp 168 is situated in β–strand 8.…”

“…Sequence alignment of the T4 gp21 protease with proteases of other tailed phages and herpesviruses (Liu and Mushegian, 2004; Thomas and Black, 2013) predicted Ser 140 and His 85 to be the gp21 residues involved in catalysis. To avoid possible self-cleavage, we produced a gp21 mutant which had alanines in positions 140 and 85.…”

Global Proteomic Profiling of Salmonella Infection by a Giant Phage

“…Based on this precedent, we hypothesized that the scaffold protein of a giant phage might also be cleaved. Since we had previously demonstrated that the KZ prohead protease gp175 is active in vitro and its cleavage specificity is known (11,35), we sought to determine whether gp54 was a substrate for gp175. The gp54 gene was cloned with the codons for an N-terminal His 6 tag.…”

Molecular architecture of tailed double-stranded DNA phages

“…For example, the core of the bacteriophage T4 prohead contains, in addition to ~580 copies of the scaffolding protein, 7 extra proteins with copy numbers ranging from 40 to 370. 13,22 Procapsids of many phages (like T4, HK97, φKZ, and λ) contain head maturation proteases (serine proteases in case of T4 13,22 and φKZ 23 ) which are activated after prohead assembly and degrade the internal core partially or completely. The digestion products exit the prohead, freeing the space for the genomic DNA.…”

“…148 Despite the low sequence similarity, these proteins have similar structures, indicating that the sheath proteins of tailed phages have evolved from a common ancestor. In the DSY3957 crystal structure the N-terminal arm (residues [15][16][17][18][19][20][21][22][23][24][25] of the protein is a part of a β-sheet in the C-terminal domain of a symmetry related molecule. Based on this observation, Leiman and Shneider 128 proposed that in the phage sheaths such chain swapping cross-links neighboring subunits stacked on top of each other, thus helping to maintain the sheath integrity.…”