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

Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gramnegativ e bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoc a pullulanase secretion pathway showed that it consists of an N-terminal do main and a protease-resistant C-terminal domain that remains multimeric aft er 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 pilo t protein PulS binds. Electron microscopy showed that the stable fragment p roduced by proteolysis is composed of two stacked rings that encircle a cen tral channel and that it lacks the peripheral radial spokes that are seen i n the native complex. Moreover, the electron microscopic images suggest tha t the N-terminal domain folds back into the large cavity of the channel tha t is formed by the C-terminal domain of the native complex, thereby occludi ng the channel, consistent with previous electrophysiological studies showi ng that the channel is normally closed.