Inactivation in vitro of the Escherichia coli outer membrane protein FhuA by a phage T5-encoded lipoprotein.
Bacteriophage T5-encoded lipoprotein, synthesized by infected Escherichia coli cells, prevents superinfection of the host cell by this virus. The molecular basis of its ability to inactivate the receptor of phage T5, the FhuA protein, was investigated in vitro. Fully competent T5 lipoprotein, with a His tag attached to the C-terminus, was purified in detergent solution. Coreconstitution with homogeneous FhuA protein into liposomes revealed that the lipoprotein inhibited the irreversible inactivation of phage T5 by FhuA protein. This phenomenon correlated with the inhibition of phage DNA ejection determined by fluorescence monitoring. Addition of detergent abolished the interaction between T5 lipoprotein and FhuA protein. When the signal sequence and N-terminal cysteinyl residue of the lipoprotein were removed by genetic truncation, the soluble polypeptide could be refolded and purified from inclusion bodies. The truncated lipoprotein interfered with infection of E. coli by phage T5, but only at very high concentrations. Circular dichroism spectra of both forms of T5 lipoprotein exhibited predominantly beta-structure. T5 lipoprotein is sufficient for inactivation of the FhuA protein, presumably by inserting the N-terminal acyl chains into the membrane, thus increasing its local concentration. An in vitro stoichiometry of 10:1 has been calculated for the phage-encoded T5 lipoprotein to FhuA protein complex.
Pedruzzi
I
I
Biozentrum, University of Basel, Switzerland.
Rosenbusch
J P
JP
Locher
K P
KP
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism* / Q000821:virology*; D029968:Escherichia coli Proteins; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism*; D013604:T-Phages / Q000502:physiology*; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008074:Lipoproteins; D008081:Liposomes; D011991:Receptors, Virus; D014764:Viral Proteins