title: Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy.
abstract: Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gram-negative bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N-terminal domain and a protease-resistant C-terminal domain that remains multimeric after proteolysis. The stable C-terminal domain starts just before the region in PulD that is highly conserved in the secretin superfamily and apparently lacks the region at the C-terminal end to which the secretin-specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N-terminal domain folds back into the large cavity of the channel that is formed by the C-terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.
authors.affiliation: Unité de Génétique Moléculaire, Centre National de la Recherche Scientifique URA 1773, 25 rue du Dr Roux, Institut Pasteur, 75724 Paris, Cedex 15, France. max@pasteur.fr
authors.forename: N
authors.initials: N
authors.lastname: Nouwen
authors.forename: H
authors.initials: H
authors.lastname: Stahlberg
authors.forename: A P
authors.initials: AP
authors.lastname: Pugsley
authors.forename: A
authors.initials: A
authors.lastname: Engel
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D006026:Glycoside Hydrolases / Q000378:metabolism; D007709:Klebsiella / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; C061377:PulD protein, Klebsiella pneumoniae; D006026:Glycoside Hydrolases; C100181:pullulanase