The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity

The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involve d in the translocation of proteins across the outer membrane via the type I I secretion pathway. Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells. However, overexpression of Pseudomonas alcaligenes XcpQ cou ld be achieved in the P. alcaligenes mutant strain 537. Protease protection experiments with P. alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin famil y, is largely resistant to proteinase K. This protease-resistant fragment i s embedded in the membrane and remains a stable complex, indicating that th is domain is involved in complex formation. Both the intact and the proteas e-protected XcpQ complex showed a tendency to form two-dimensional crystal- like structures. Electron microscopic analysis of these structures showed t hat the overall oligomeric rings of the intact and of the protease-resistan t complex are highly similar. The central cavity of the intact XcpQ complex contains structured mass. Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent w ith their role as a translocation channel. However, the single-channel cond uctances observed were not uniform. Together, these results demonstrate tha t the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported. (C) 1999 Academic Press.