title: Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD.
abstract: We engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced.
authors.affiliation: Unité de Biochimie Structurale, Centre National de la Recherche Scientifique URA2185 and Unité de Génétique Moléculaire, Centre National de la Recherche Scientifique URA2172, Institut Pasteur, 75724 Paris Cedex 15, France.
authors.forename: Barbara
authors.initials: B
authors.lastname: Mouratou
authors.forename: Francis
authors.initials: F
authors.lastname: Schaeffer
authors.forename: Ingrid
authors.initials: I
authors.lastname: Guilvout
authors.forename: Diana
authors.initials: D
authors.lastname: Tello-Manigne
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pecorari
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011863:Radioimmunoassay; D020349:Surface Plasmon Resonance
chemical_list: D001425:Bacterial Outer Membrane Proteins; D004268:DNA-Binding Proteins