title: Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD.
abstract: The DNA-binding protein Sac7d was previously modified to bind with high affinity to the N domain of the outer membrane secretin PulD from the bacterium Klebsiella oxytoca. Here, we show that binding of the Sac7d derivatives (affitins) to PulD is sensitive to conformational changes caused by denaturant and by the zwitterionic detergent Zwittergent 3-14 routinely used to extract secretins from outer membranes. This sensitivity to the conformational state of PulD allowed us to use the affitins as probes for the native structure of PulD and to devise protocols for examining in vitro synthesized protein in nonionic detergent and for the affinity purification of native PulD using affitins as ligands. When fused to periplasmic PhoA, three affitins inhibited PulD multimerization in vivo and caused loss of function. In two cases, this was likely to be due to dimerization of the affitin by the bound PhoA, as the effect was absent when the affitins were fused to monomeric MalE. In the third case, the MalE and PhoA moieties probably interfered sterically with PulD protomer interactions and, thereby, inhibited multimerization. None of the affitins tested interacted with PulD at sites of protomer interaction or blocked the secretin channel through which exoproteins cross the outer membrane in the Type II secretion pathway of which PulD is a key component.
authors.affiliation: Institut Pasteur, Unité de Génétique moléculaire, CNRS URA2172, 25, rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Martin
authors.initials: M
authors.lastname: Krehenbrink
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.forename: Ingrid
authors.initials: I
authors.lastname: Guilvout
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pécorari
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D000939:Epitopes / Q000737:chemistry; D041121:Klebsiella oxytoca / Q000737:chemistry*; D008854:Microscopy, Electron; D015335:Molecular Probes / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D012633:Secretin / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D003902:Detergents; D000939:Epitopes; D015335:Molecular Probes; D010455:Peptides; D012633:Secretin; C095116:Sac7 protein, Sulfolobus; D014508:Urea; D017830:Octoxynol