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EMBO J. 2000 May 15;19(10):2229-36.

Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy.

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1
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

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.

PMID:
10811614
PMCID:
PMC384361
DOI:
10.1093/emboj/19.10.2229
[Indexed for MEDLINE]
Free PMC Article
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