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Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy
Author(s) -
Nouwen Nico,
Stahlberg Henning,
Pugsley Anthony P.,
Engel Andreas
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.10.2229
Subject(s) - biology , proteolysis , secretin , electron microscope , domain (mathematical analysis) , biophysics , computational biology , biochemistry , enzyme , pancreas , mathematical analysis , physics , mathematics , optics
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.