IADR Abstract Archives
Control of Bacterial Sex Pheromone Activity by PrgY: a Structural Homolog of a Metalloprotease (TIKI) that modulates Wnt Signaling in Eukaryotes
Objectives: The chromosomally encoded pheromone cCF10 induces conjugative transfer of Enterococcus faecalis plasmid pCF10. The pCF10 PrgY protein reduces production of endogenous pheromone by donor cells to prevent self-induction. PrgY-like proteins are found in all biological kingdoms, yet little is known about the functional mechanism of this family of proteins. The goal is to study the biochemical activity of PrgY.
Methods: We used pull-down assays and Surface Plasmon Resonance (SPR) to demonstrate direct, specific interaction, and binding affinity between PrgY and cCF10. Mass spectrometry is being used currently to examine whether PrgY-dependent cCF10 degradation products can be detected in donor culture supernatants, or following incubation of cCF10/PrgY mixtures in vitro.
Results: Strong binding between cCF10 and PrgY was observed and the binding of cCF10 to PrgY can be saturated at a level comparable to the calculated theoretical maximum (60RU) assuming 1:1 binding. The binding affinity of a synthetic cCF10 variant (L4I) with reduced in vivo susceptibility to PrgY is lower than cCF10 but can still be saturated at 60RU. No significant binding was observed between a PrgY mutant (R50Q) that fails to reduce cCF10 production in vivo. PrgY shares significant homology to the eukaryotic metalloprotease TIKI that regulates the Wnt signaling. Using a structural prediction algorithm, Bazan et al showed that the two proteins share key conserved residues in the metal-binding catalytic core as well as overall secondary structure. Four key conserved residues in the putative active site of TIKI are H39, E66, E140, and H311 and the corresponding conserved residues in PrgY are H21, E45, D120, and H215 respectively.
Conclusions: These data support part of our hypothetical model for control of endogenous pheromone in donor cells by PrgY.
Methods: We used pull-down assays and Surface Plasmon Resonance (SPR) to demonstrate direct, specific interaction, and binding affinity between PrgY and cCF10. Mass spectrometry is being used currently to examine whether PrgY-dependent cCF10 degradation products can be detected in donor culture supernatants, or following incubation of cCF10/PrgY mixtures in vitro.
Results: Strong binding between cCF10 and PrgY was observed and the binding of cCF10 to PrgY can be saturated at a level comparable to the calculated theoretical maximum (60RU) assuming 1:1 binding. The binding affinity of a synthetic cCF10 variant (L4I) with reduced in vivo susceptibility to PrgY is lower than cCF10 but can still be saturated at 60RU. No significant binding was observed between a PrgY mutant (R50Q) that fails to reduce cCF10 production in vivo. PrgY shares significant homology to the eukaryotic metalloprotease TIKI that regulates the Wnt signaling. Using a structural prediction algorithm, Bazan et al showed that the two proteins share key conserved residues in the metal-binding catalytic core as well as overall secondary structure. Four key conserved residues in the putative active site of TIKI are H39, E66, E140, and H311 and the corresponding conserved residues in PrgY are H21, E45, D120, and H215 respectively.
Conclusions: These data support part of our hypothetical model for control of endogenous pheromone in donor cells by PrgY.