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PMID:16970964

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Citation

Olia, AS, Al-Bassam, J, Winn-Stapley, DA, Joss, L, Casjens, SR and Cingolani, G (2006) Binding-induced stabilization and assembly of the phage P22 tail accessory factor gp4. J. Mol. Biol. 363:558-76

Abstract

To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.

Links

PubMed Online version:10.1016/j.jmb.2006.08.014

Keywords

Bacteriophage P22/chemistry; Bacteriophage P22/metabolism; Binding Sites; Models, Molecular; Protein Binding; Protein Conformation; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Salmonella typhimurium/virology; Temperature; Time Factors; Viral Tail Proteins/chemistry; Viral Tail Proteins/genetics; Viral Tail Proteins/metabolism; Viral Tail Proteins/ultrastructure

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

BPP22:EXLYS

GO:0098015: virus tail

ECO:0000314:

C

Figure 9. Negative stain electron microscopy of tail accessory factor gp4 bound to P22 portal protein rings. (a) Negative stain electron microscopy image of purified P22 portal protein rings. Many “donut-like” structures are visible, which in most (rarer) side view cases adopt a head-to-head conformation. (b) Negative stain micrograph of gel filtration purified portal protein in complex with gp4.

Micrographs of the gp1:gp4 complex were compared to gp4-free portal rings, as shown in Figure 9(a) and (b), respectively. The majority of gp1:gp4 complexes seen on the micrographs displayed a preferential orientation on the grid with the central hole perpendicular to the grid. In rare instances single complexes and head-tohead dimers of the gp1:gp4 complex were seen in side view (see higher magnifications in Figure 9(b))

complete
CACAO 12028

BPP22:EXLYS

GO:0046798: viral portal complex

ECO:0000314:

C

Figure 7. Isolating the gp1:gp4 assembly intermediate on agarose gel. (a) Native agarose gel run at 30 °C showing a stably populated gp(1)12:gp(4)6 assembly intermediate. In lane 1 is dodecameric portal protein gp(1)12. The gp(1)12:gp(4)6 assembly intermediate in lanes 2 and 3 migrates on gel as a slightly lower mobility band, clearly distinguishable from fully saturated decorated gp(1)12:gp(4)12 complex in lane 4 and free dodecameric portal protein gp(1)12 in lane 4. The intermediates in lanes 2 and 3 were formed by adding six equivalents of gp4 to gp(1)12 and incubating the complex at 30 °C for 30 s and 30 min, respectively. In lane 4 approximately eight equivalents of gp4 were added, yielding the fully decorated gp(1)12:gp(4)12 complex and free gp(1)12. (b) Titration of gp4 binding to gp1 at 30 °C. By running the agarose gel at 30 °C both gp(1)12:gp(4)6 assembly intermediate and fully decorated gp(1)12:gp(4)12 portal protein are visible on the same titration. The assembly intermediate appears at stoichiometries gp(1):gp(4) equal to 12 (lanes 3−6) and fades away in lane 7, where >six equivalents of gp4 are present.

Micrographs of the gp1:gp4 complex were compared to gp4-free portal rings, as shown in Figure 9(a) and (b), respectively. The majority of gp1:gp4 complexes seen on the micrographs displayed a preferential orientation on the grid with the central hole perpendicular to the grid. In rare instances single complexes and head-tohead dimers of the gp1:gp4 complex were seen in side view (see higher magnifications in Figure 9(b)). Such side views are particularly informative, in that the dodecameric portal protein without bound gp4 forms a mushroom-shaped structure with a channel through the center

complete
CACAO 12031

Notes

See also

References

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