title: An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.
abstract: The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.
authors.affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.
authors.forename: Hyeon-Cheol
authors.initials: HC
authors.lastname: Lee
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853 USA.
authors.forename: Alyse D
authors.initials: AD
authors.lastname: Portnoff
authors.affiliation: Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853 USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rocco
authors.affiliation: 1] School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA [2] Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853 USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: DeLisa
mesh_terms: D042931:Aldehyde Oxidase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D020836:Protein Structure, Quaternary
chemical_list: D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D042931:Aldehyde Oxidase