The T7 ejection nanomachine components gp15–gp16 form a spiral ring complex that binds DNA and a lipid membrane

Abstract

Bacteriophage T7 initiates infection by ejecting several internal capsid proteins into the host cell; these proteins then assemble into a nanomachine that translocates the viral genome from the phage head into the cytoplasm. The ejected proteins are thought to partially unfold as they pass through the lumen of the portal and the short stubby T7 tail during their entry into the cell. In vivo, the internal proteins gp15 and gp16 assemble into a tubular structure that spans the periplasm and cytoplasmic membrane. We show here that purified gp15 and gp16 can refold from a partially denatured state in vitro, and that gp15 interacts with gp16 to form a spiral ring structure. Purified gp15 binds to DNA, whereas gp16 binds protein-free liposomes; the gp15-gp16 complex binds both DNA and liposomes. Limited proteolysis of the liposome-bound gp16 reveals that its C-terminal region is protected, suggesting a partial membrane insertion of the protein.

T7 ejectosome assembly: A story unfolds

“…6 We observed that the mainly a-helical gp15 partially unfolds at temperatures as low as 458C with an unfolding transition point of 498C, and destabilization of gp16 is observed at 408C with a thermal transition point of 44 8C. These temperatures indicate that only small amounts of energy are required for the unfolding of both proteins.…”

“…We recently demonstrated that gp15 and gp16 have the propensity to regain their structure after being fully thermally unfolded. 6 Here, we extended this work and show that gp15 is chemically labile and unfolds in the presence of low concentrations of guanidine hydrochloride. We also show that the gp15-gp16 complex binding to liposomes coincides with a small but significant increase of a-helical structure.…”

“…While it is not known whether gp15 and gp16 are spheroid in shape, size exclusion chromatography of gp15 shows the typical behavior of a globular shaped protein, while gp16 does not. 6 Therefore, it is a fair assumption that at least gp15 cannot translocate in its folded structure and most definitely not as a pre-formed, folded complex with gp16. Certainly, oligomeric assemblies of both proteins cannot pass through the central cavity of the portal.…”

“…2C), as they form a heterotrimeric complex with with two gp15 and one gp16 subunit. 6 The peptidoglycan hydrolase activity of gp16 allows penetration of the periplasmic space and assembly of a hollow tube extending toward the inner membrane. In the final assembly step of the ejectosome, the proteins eventually form a (trans-) membrane complex (Fig.…”

“…Evidence for this last step is our observation that the gp15-gp16 complex binds to lipid membranes mediated by gp16, as this protein interacts with liposomes on its own. 6 Whether or not the complex also creates a pore in the membrane or whether it hijacks an inner-membrane host protein 2 is currently unanswered. In order to investigate how the ejectosome proteins gp15-gp16 bind to liposomes, we analyzed their structure in the complex in presence and absence of POPE:POPG (70 mol % and 30 mol %, respectively).…”